Artificial Transcription Factors and Uses Thereof

ABSTRACT

The present invention relates to artificial transcription factors (ATFs) that alter gene expression, including inducing pluripotency in cells or promoting the conversion of cells to specific cell fates. In particular, provided herein is a zinc-finger based ATF library that can be screened in cells by looking for expression of a specific gene (e.g., reporter expression), monitoring for cell surface markers or morphology, or via functional assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/425,374, filed Nov. 22, 2016, which is incorporated herein by reference as if set forth in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under HL099773 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Artificial transcription factors (ATFs) are DNA-binding molecules designed to control gene expression in a pre-determined manner. Rather than taking the conventional approach of testing candidate factors curated from studying embryonic development or differential expression analysis, screening a gene-activating ATF library can be a highly effective and orthogonal approach to sample thousands of sites in parallel and activate a cell fate-defining transcriptional network. An ATF library can sample concurrently the level of activation for thousands of sites because each ATF has different affinity and accessibility to DNA. Because ATFs do not rely on endogenously expressed co-factors and are not restrained to feedback circuits like natural factors, they can serve as powerful agents to perturb the homeostatic state of any cell type. The target genes of the ATFs can then be used to understand the mechanism of action driving the phenotypic change or cell fate conversion.

Transcription factors are modular by nature, and each domain can be tailored to create ATFs for programmed regulation of genes and networks (Eguchi et al., Biochemical Journal 462(3):397-413 (2014)). The DNA-binding domain (DBD) confers sequence specificity in targeting genomic loci. The effector domain provides the ATF with function, be it transcriptional activation, repression, or modification of chromatin. Additionally, an interaction domain can be incorporated in the design such that the ATF can interact with other factors in the cell (Id.).

Among the many DBDs available to design an ATF (e.g., CRISPR/Cas9, TAL-effectors, and polyamides), zinc fingers have high affinity to DNA and can be potent regulators of transcription when fused to the appropriate effector domain. Compared to zinc fingers and TAL-effectors that can upregulate genes to biologically-relevant levels (Bailus et al., 2016; Gao et al., 2013; Rebar et al., 2002), the magnitude of transcriptional change induced by nuclease-dead CRISPR/Cas9 systems with a single guide is not as robust (Esvelt et al., 2013). Recent modifications to the CRISPR/Cas9 system have improved their impact on the level of expression of target genes; however, these modifications come at the expense of increasing their size (Chavez et al., 2015) or introducing additional effector molecules that can be recruited by Cas9 (Gilbert et al., 2014; Tanenbaum et al., 2014) or the guide RNA (Konermann et al., 2015; Zalatan et al., 2015). Unlike zinc fingers and TAL-effectors, the CRISPR/Cas9 system also requires at least two components, Cas9 and the guide, to be delivered to the same cell. Polyamides, a class of small molecule DBDs, are not genetically encoded; rather, they are added to the media and rely on cell uptake. However, the rules governing polyamide permeability are still not well understood, making delivery to cells problematic (Edelson et al., 2004).

Accordingly, there remains a need in the art for improved artificial transcription factors and efficient protocols for generating pluripotent stem cells and differentiated cell types under controlled conditions.

BRIEF SUMMARY

In a first aspect, provided herein is an artificial transcription factor comprising a polydactyl zinc finger protein comprising two or three zinc finger domains, an interaction domain, an optional nuclear localization signal, and an activation domain, where each of the two or three zinc finger domains comprises a variable domain independently encoded by nucleic acid sequence of SEQ ID NO:2 (VNN-TCC-VNN-VNN-CTC-ACC-VNN), where each VNN of SEQ ID NO:2 is a codon corresponding to an amino acid selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V. The polydactyl zinc finger protein can comprise three zinc finger domains encoded by SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, where each VNN of SEQ ID NOS:4, 5, and 6 is a codon corresponding to an amino acid selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V. The interaction domain can comprise a 15-amino acid peptide that enables interaction with the hydrophobic face of the most 5′ zinc finger domain. The activation domain can comprise four tandem repeats of DALDDFDLDML (SEQ ID NO:7). The nuclear localization signal can be encoded by an amino acid sequence selected from the group consisting of KDKKADKSVV (SEQ ID NO: 11) and PKKKRKV (SEQ ID NO:12).

In another aspect, provided herein is a method of reprogramming a somatic cell to pluripotency, wherein the method comprises (a) exposing a somatic cell to a plurality of artificial transcription factors, wherein the artificial transcription factors are selected from the group consisting of ZFATF1, ZFATF2, ZFATF3, ZFATF4, and ZFATF5; (b) further exposing the somatic cell to a plurality of potency determining factors comprising Sox2, Klf4, and c-Myc; and (c) culturing the exposed cells to obtain reprogrammed cells having a higher potency level than the somatic cell. The plurality of ATFs can comprise ZFATF1, ZFATF2, and ZFATF5. The plurality of ATFs can comprise ZFATF1, ZFATF2, and ZFATF4. The plurality of ATFs can comprise ZFATF1, ZFATF2, and ZFATF3. The plurality of ATFs can comprise ZFATF1 and ZFATF4.

In another aspect, provided herein is a method of directing differentiation of a pluripotent stem cell to a cardiomyocyte. The method can comprise or consist essentially of (a) exposing a pluripotent stem cell to one or more artificial transcription factors (ATFs) selected from the group consisting of ZFATF1, ZFATF2, and ATF5; and (b) culturing the exposed cells of (a) in the presence of a Wnt activator for about 7-10 days, such that a population of cells comprising cardiomyocytes is obtained. The ATFs can comprise ZFATF1, ZFATF2, and ATF5. The Wnt activator can be a GSK3 inhibitor. The GSK3 inhibitor can be CHIR99021.

In a further aspect, provided herein is a method of directing differentiation of a pluripotent stem cell to a hematopoietic lineage. The method can comprise or consist essentially of (a) exposing a pluripotent stem cell to two or more artificial transcription factors (ATFs) selected from the group consisting of ZFATF19, ZFATF20, ZFATF21, ATF5, ZFATF1, ZFATF6, ZFATF10, ZFATF13, ZFATF17, ZFATF22, ZFATF23, ZFATF24, ZFATF25, ZFATF26, ZFATF27, ZFATF28, ZFATF29, ZFATF30, and ZFATF31; and (b) culturing the exposed cells of (a) in a basal culture medium comprising FGF2, SCF, and thrombopoietin, and in the presence of TAL for about 7-10 days, such that a cell population comprising hematopoietic lineage cells is obtained. The ATFs can comprise ZFATF19, ZFATF20, ZFATF21, and ATF5, and wherein the cell population comprises erythroid-like cells. The ATFs can comprise ZFATF1, ZFATF6, ZFATF10, ZFATF13, ZFATF17, ZFATF22, ZFATF23, ZFATF24, ZFATF25, ZFATF26, ZFATF27, ZFATF28, ZFATF29, ZFATF30, and ZFATF31, and wherein the cell population comprises monocyte-like cells.

In a further aspect, provided herein is a method for preparing a zinc finger-based artificial transcription factor (ATF) library comprising a plurality of elements, whereby each element of said ATF library comprises a zinc finger backbone comprising two or three oligonucleotides, each encoding SEQ ID NO:3 (X⁻¹-S-X₂-X₃-L-T-X₆), an interaction domain, an optional nuclear localization signal, and an activation domain, wherein X⁻¹ is an amino acid preferably selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V; X₂ is preferably selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V; X₃ is preferably selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V; and X₆ is preferably selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V. The ATF library can have a complexity of about 2.62×10⁶. The interaction domain can comprise a 15-amino acid peptide that enables interaction with the hydrophobic face of the most 5′ zinc finger domain. The activation domain can comprise four tandem repeats of DALDDFDLDML (SEQ ID NO:7). The nuclear localization signal can be encoded by an amino acid sequence selected from the group consisting of KDKKADKSVV (SEQ ID NO:11) and PKKKRKV (SEQ ID NO:12).

In a further aspect, provided herein is a zinc finger-based artificial transcription factor (ATF) library obtained according to methods provided herein.

These and other features, objects, and advantages of the present invention will become better understood from the description that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of the invention. The description of preferred embodiments is not intended to limit the invention to cover all modifications, equivalents and alternatives. Reference should therefore be made to the claims recited herein for interpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The present invention will be better understood and features, aspects, and advantages other than those set forth above will become apparent when consideration is given to the following detailed description which makes reference to the following drawings, wherein:

FIGS. 1A-IF illustrate exemplary ATFs designed with three zinc fingers, activation domain, and interaction domain to maximize transcriptional effects. (A) Architecture of the ATF. From N to C terminus, the ATF consists of an interaction domain (ID), three zinc fingers, a nuclear localization signal (NLS), and a VP64 activation domain. The interaction domain is a 15-amino acid peptide that enables interaction with the hydrophobic face of the first finger. The palindromic EGR1 binding site 5′-GCG-TGG-GCG-CGC-CCC-CGC-3′ (SEQ ID NO: 1) was cloned upstream of a luciferase reporter for the assay in (B). (B) ATF with three zinc fingers, an interaction domain, nuclear localization signal, and a VP64 activation domain has highest level of induction. The DNA binding domain of human EGR1 was tested with and without an ID, NLS, and VP64 in the expression of a luciferase reporter in HEK293 cells (n=4, p <0.05 by one-way ANOVA with post-hoc Tukey test). (C) Specificity and Energy Landscapes (SELs). SELs display the comprehensive binding preferences of a transcription factor based on a chosen seed motif. The height of the peak indicates higher relative affinity for a given sequence. Sequences, which are one or two base pairs longer than the seed motif, are arranged in concentric rings with respect to mismatches to the seed motif. Within the mismatch rings, the sequences are arranged by position of the mismatch, then alphabetically by the actual sequence. For example, the teal section labeled with the curved arrow displays all the sequences with a mismatch at the second position. (D) The interaction domain allows the ATF to bind a greater number of sequences. The DNA binding domain of EGR1 fused to an ID binds sites recognized by a dimer, which is not recognized by a monomer. The SEL displayed shows data after three rounds of enrichment. (E) RNA-seq results show that the 3-zinc finger ATF with an ID upregulates transcription of the largest number of genes compared to the mock transfected control. Among the upregulated genes, 18 genes are expressed 50-fold or more by the 3-zinc finger ATF with an ID. All ATFs in this assay have an NLS and VP64 (n=1, p<0.0005). (F) Design of the ATF library. The residues that confer specificity (−1, 2, 3, and 6 positions) were randomized to amino acids represented by VNN codons. A library with a complexity of 2.6×10⁶ members was created. The zinc finger scaffold comes from human EGR1 and is fused to the ID described in (A), NLS, and VP64. Sequencing of 100 ATFs from the library shows all 16 amino acids represented by the recognition residues. This sample size is representative of the library at a 95% confidence interval and a margin of error of 9.8%.

FIGS. 2A-2E demonstrate an application of the ATF library in induction of pluripotency. (A) Genetic screen with an ATF library. (1) The ATF library was cloned into pSIN, a second generation lentiviral vector. The ATF is driven by a constitutive promoter, EF1α. The puromycin resistance gene enables selection of cells with integration events. The screen can be performed on cells with a robust change in phenotype, such as the upregulation of a cell surface marker or a distinct change in morphology or function. Alternatively, a lineage-specific reporter system can be used to identify hits, or successful cell fate conversions. (2) Hits from the library are isolated as single cells, such that combinations of ATFs, if any, can be captured. (3) Integrated ATFs are identified from single cells by two-step nested PCR. (4) Identified ATFs are retested to validate whether they recapitulate the phenotype seen in the original screen. Once validated, RNA-seq can be performed to determine how the ATFs change the expression profile of the cell. Additionally, ChIP-seq can be performed to determine the direct genomic targets. (B) Testing the ATF library in mouse embryonic fibroblasts (MEFs) bearing a reporter for Oct4. These MEFs have a Tomato red reporter gene at the ROSA locus. Upon induction of Oct4, a tamoxifen-inducible Cre recombinase (MerCreMer) is co-expressed. The addition of 4-hydroxy-tamoxifen to the system results in the excision of Tomato red, which allows for the expression of GFP, thereafter. (C) Flow cytometry results at Day 15 after introduction of transcription factors. Tomato⁻GFP⁺ MEFs transduced with the ATF library, Sox2, Klf4, and c-Myc were isolated as single cells for further analysis. MEFs treated with Oct4, Sox2, Klf4, and c-Myc (OSKM) were used as a positive control. Untreated MEFs served as a negative control. The percentage of Tomato⁻GFP⁺ (Q3) was higher in the ATF library treated cells when compared to the OSKM control. Cells, which were double positive (Tomato⁺GFP⁺; Q2), were also collected as those in which Oct4 was expressed later than the Tomato⁻GFP⁺ cells (Q3). Percentages in each quadrant are displayed under the quadrant number. (D) ATFs were identified from eleven single cells from (C) by two-step nested PCR of genomic DNA. Unique ATFs are depicted with a different color. One ATF had a frameshift mutation shortly after the ID, coding for a protein, which does not have a zinc finger structure. A few ATFs, notably the blue and orange ATFs, are expressed in most of the cells analyzed. The ATF with the frameshift mutation is also expressed in many of the hits. All cells except #4 were collected as Tomato⁻GFP⁺ cells. Cell #4 was Tomato⁺GFP⁺ at Day 15. (E) Three combinations of ATFs (#2, #3, and #4) successfully replaced Oct4 in inducing pluripotency with Sox2, Klf4, and c-Myc. Micrographs of MEFs before induction of exogenous factors are Tomato⁺GFP⁻. iPS cells generated with ATFs are similar to those generated with OSKM and are Tomato⁻GFP⁺. Two ATFs in each combination are the same (blue and orange). Bars=100 μm.

FIGS. 3A-3D demonstrate that iPS (induced pluripotent stem) cells generated with ATFs are pluripotent. (A) Immunofluorescence staining of iPS colonies with OCT4, SOX2, and NANOG. These iPS cells were generated with Combination #2+SKM. Bars=100 μm. (B) Embryoid bodies were cultured with iPS cells generated with Combination #2+SKM. Micrographs were taken at Day 2 and Day 14 of differentiation. Immunofluorescence staining for MYL2 (mesoderm), FOXA2 (endoderm), and TUBB3 (ectoderm) of embryoid body outgrowths show potential to differentiate into all three germ layers. Each micrograph was also stained with DAPI to label nuclei. Bars=100 μm. (C) Embryoid bodies derived from Combination #2+SKM iPS cells differentiate into all three germ layers. RT-qPCR results from RNA harvested from ATF-generated iPS cells and embryoid bodies derived from them. Markers for mesoderm are brachyury (T), NK2 homeobox 5 (Nkr2-5), and kinase insert domain protein receptor (Kdr). Markers for endoderm are alpha fetoprotein (Afp), transthyretin (Ttr), and forkhead box A2 (Foxa2). Markers for ectoderm are nestin (Nes), neurofilament, light polypeptide (Nefl), and SRY-box 17 (Sox17). Values are log₂(ΔΔCt+1)±SEM relative to Gapdh (n=3). A Student's two-tailed t-test was used to determine p values. A p value of <0.1 was considered significant. (D) Data points for RT-qPCR results described in (C). Values are log₂(ΔΔCt+1) relative to Gapdh (n=3).

FIGS. 4A-4H demonstrate that iPS cells generated with ATFs are pluripotent. (A) Immunofluorescence staining of C2+SKM iPS colonies with OCT4, SOX2, and NANOG. Bars=100 μm. (B) Teratoma assay results show differentiation into mesoderm, endoderm, and ectoderm. Bar=100 μm. (C) iPS cells generated with ATFs cluster with mouse ES cells and iPS cells generated with Oct4+SKM. Samples marked early are MEFs transduced with the indicated factors before conversion into iPS cells between Day 18-27 (n=3 or 4). (D) A heatmap of fibroblast and pluripotency markers of iPS cells generated with ATFs shows downregulation of fibroblast genes and upregulation of pluripotency genes. Scale displays differential expression log₂(ratio relative to mean). (E) A heatmap of 853 genes from the CellNet fibroblast gene regulatory network (GRN) show iPS cells generated with ATFs obtain transcriptional profiles similar to that of other pluripotent cells (Oct4+SKM iPS cells and ES cells). Scale displays differential expression log₂(ratio relative to mean). (F) A heatmap of 705 genes from the CellNet pluripotency GRN show iPS cells generated with ATFs have expression profiles similar to that of other pluripotent cells. Scale displays differential expression log₂(ratio relative to mean). (G) H3K27ac marks, specifying active regions of chromatin, appear in a common set of genes for Oct4+SKM iPS cells, C2+SKM iPS cells, and C3+SKM iPS cells. H3K27ac peaks were annotated to genes with Homer to create Venn diagrams for genes. ChIP enrichment among treatments was determined to be significant at an FDR <0.1 by DiffBind (n=2). (H) H3K9me3 marks, specifying repressed regions of chromatin, appear in a common set of genes for Oct4+SKM iPS cells, C2+SKM iPS cells, and C3+SKM iPS cells. H3K9me3 peaks were annotated to genes with Homer to create Venn diagrams for genes. ChIP enrichment among treatments was determined to be significant at an FDR <0.1 by DiffBind (n=2).

FIGS. 5A-5C demonstrate that iPS cells generated with ATFs have similar sets of active histone marks as iPS cells generated with Oct4, Sox2, Klf4, c-Myc (“OSKM”). The genome-wide chromatin modification landscapes in ATF-induced iPS cells were compared to iPS cells generated with Oct4+SKM. Specifically, ChIP-seq was performed on histone 3 lysine 27 acetylation (H3K27ac), the marker delineating active promoters and super-enhancers that define cell identity (Whyte et al., 2013; Hnisz et al., 2013), and histone 3 lysine 9 trimethylation (H3K9me3), the marker that is strongly correlated to repressed regions of the genome that are bound by heterochromatin protein 1 (Schultz et al., 2002). (A) A large fraction of H3K27ac marks, an active mark for super-enhancers, is shared among OSKM iPS cells, Combination #2+SKM iPS cells, and Combination #3+SKM iPS cells. MEFs exhibit a unique set of H3K27ac peaks compared to iPS cells in this study (MEF data from Mouse ENCODE). ChIP enrichment among treatments was determined to be significant at an FDR <0.1 by DiffBind (n=2). ChIP traces over a 5 kb window for representative examples are shown for the Venn diagram categories. (B) Peaks were annotated to genes with Homer to create Venn diagrams for genes. (C) Spearman correlation shows that iPS cells correlate better with each other than to MEFs.

FIGS. 6A-6C demonstrate that iPS cells generated with ATFs have similar sets of repressive histone marks as iPS cells generated with Oct4, Sox2, Klf4, c-Myc (“OSKM”). (A) A subset of H3K9me3 marks, a marker of repressed loci, is shared among OSKM iPS cells, Combination #2+SKM iPS cells, and Combination #3+SKM iPS cells. More peaks are shared between OSKM iPS cells and Combination #3+SKM iPS cells. ChIP enrichment among treatments was determined to be significant at an FDR <0.1 by DiffBind (n=2). ChIP traces over a 5 kb window for representative examples are shown for the overlapping Venn diagram categories. (B) Peaks were annotated to genes with Homer to create Venn diagrams for genes. (C) Spearman correlation shows that Combination #3+SKM iPS cells correlate slightly better to OSKM iPS cells than do Combination #2+SKM iPS.

FIGS. 7A-7C demonstrate that ATFs activate key regulators of the pluripotency network. (A) Workflow for determining ATF target genes for C2+SKM. Three pairwise comparisons were made: 1) C2+SKM iPS cells vs Empty+SKM cells, 2) C2+SKM early cells vs Empty+SKM cells, 3) C2+SKM iPS cells vs Oct4+SKM iPS cells. Genes upregulated >2-fold (p<0.05) in the cells transduced with ATFs with ATF binding sites within a ±1 kb window of the TSS were determined to be potential targets. Binding sites were identified by using the top 5 scoring 10-bp motifs from CSI. These target genes were used to build the network in FIG. 6A with information from the literature and the STRING database. (B) Differentially expressed pluripotency genes with ATF binding sites within ±1 kb of the transcriptional start site (TSS). ATF binding sites were derived from the top 5 scoring 10-bp motifs from CSI for C2+SKM. (C) ChIP-seq signal for HA tag on ATFs for five predicted targets in FIG. 5B. Additional ChIP-seq traces are in Fig. S5A. Traces display total reads for C2+SKM and Empty+SKM cells at an intermediate stage before reprogramming to a pluripotent state.

FIGS. 8A-8F demonstrate that the three zinc finger design has greatest impact on expression profile. (A) The three-zinc finger ATF induces expression 329-fold over the mock control, while the two-zinc finger ATF induces expression 2-fold in a luciferase assay performed in HEK293 cells. The DNA-binding domain comes from either the first two or all three zinc fingers of EGR1. Each ATF is also comprised of an ID, NLS, and VP64 (n=4, p<0.01 by one-way ANOVA with post-hoc Tukey test). (B) Cognate site identification (CSI). This in vitro method of determining the sequence specificity of DNA-binding factors involves incubating the transcription factor (TF) with a randomized library of 25-bp sequences. The TF-DNA complexes are captured with an antibody, and bound DNA is PCR-amplified for the next round of selection. Three rounds of selection are performed, and all three rounds are sequenced in a high-throughput manner to obtain Specificity and Energy Landscapes (SELs). Data obtained from sequencing can be used to generate position weight matrices. (C) The binding specificity of the first two zinc fingers of EGR1 changes without the third zinc finger. The first two zinc fingers of EGR1 bind 5′-TGG-GCG-3′; however, in the absence of the third zinc finger, the cognate site becomes 5′-GGG-ATC-3′. The interaction domain alters the binding preferences slightly, conferring greater specificity. The SEL displays data from three rounds of enrichment. (D) The binding specificity of the three zinc fingers of EGR1 is nearly identical when the seed motif is the 9-bp cognate site. The SEL displays data after three rounds of enrichment. (E) RNA-seq of HEK293 cells transfected with various ATFs show that the three-zinc finger ATF with an ID activates the greatest number of genes (n=1, p<0.0005). The heatmap displays 174 differentially regulated genes. The four different ATFs are: two-zinc fingers with and without an ID and three-zinc fingers with and without an ID. The DNA-binding domain is derived from EGR1, which is the first labeled row on the heatmap to highlight that the ATFs are overexpressed. All ATFs have an NLS and a VP64 activation domain. (F) Comparison of the number of differentially expressed genes when introduced with an ATF shows that the three-zinc finger ATF with the interaction domain affects the greatest number of genes (fold change >2, p<0.0005).

FIGS. 9A-9D demonstrate that iPS lines expanded from the ATF screen express pluripotency markers. (A) Conditions for the ATF library screen. Oct4, Sox2, Klf4, and c-Myc were delivered by retrovirus, where indicated. Second generation lentiviruses (highlighted in blue) were used to deliver the ATF library and an empty ORF control to account for false positive events that may arise from integration of a strong constitutive promoter at relevant genomic sites. Puromycin selection was performed for two days in MEFs treated with lentiviruses to select for integration events. After 15 days of culture in ES cell media, the Tomato⁻GFP⁺ cells were isolated by flow cytometry. (B) Homologous region between the ATF with the frameshift mutation and zinc finger protein 3-like from Gorilla gorilla is C-terminal to the DNA-binding domain. The G. gorilla protein (blue) contains 34 amino acids that match the ATF with the frameshift mutation (red). Within the homologous region, there are 10 amino acids, which contain similar side chains (+). Highlighted in yellow are the five zinc fingers of the G. gorilla protein. The position at which the single nucleotide frameshift mutation occurs in the ATF is indicated with a red arrow (after 34th amino acid). (C) RT-qPCR results of pluripotency markers, Oct4, Sox2, Nanog, Lin28a, Esrrb, and Utf1, in colonies of iPS cells generated with the ATF library+SKM (LSKM) show comparable levels of endogenous gene expression when compared to levels in mouse ES cells and iPS cells generated with OSKM. These colonies are derived from iPS cells expressing mixed combinations of ATFs. For each gene, transcript levels for MEFs are set to 1. Transcript levels are normalized to Gapdh. (D) Doubling time in hours of iPS cells generated with OSKM and ATFs+SKM. Values are mean±SEM (n=3).

FIGS. 10A-10E demonstrate functional pluripotency of C3+SKM and C4+SKM iPS cells. (A) Immunofluorescence staining of iPS colonies with OCT4, SOX2, and NANOG. Bars=100 μm. (B) Embryoid bodies were cultured with iPS cells generated with C3+SKM or C4+SKM. Micrographs were taken at Day 2 and Day 14 of differentiation. Bars=100 μm. (C) Immunofluorescence staining for MYL2 (mesoderm), FOXA2 (endoderm), and TUBB3 (ectoderm) of embryoid body outgrowths on Day 14 of differentiation. DAPI labels nuclei. Bar=100 μm. (D) Percentage of positive cells in (C). (E) Embryoid bodies derived from C3+SKM or C4+SKM iPS cells differentiate into all three germ layers. RT-qPCR results from RNA harvested from ATF-generated iPS cells and embryoid bodies derived from them. Markers for mesoderm (T, Nkx2-5, and Kdr) were measured at Day 7. Markers for endoderm (Ttr, Afp, and FoxA2) were measured on Day 11. Markers for ectoderm (Nes, Nefl, and Sox17) were measured on Day 14. Data points represent fold-change over iPS cells of ΔΔCt relative to Gapdh (n=3).

FIGS. 11A-1C present results of RNA-Seq analysis. (A) Clustergram of replicates generated from genome-wide RNA-seq data of the following conditions: mouse embryonic fibroblasts (MEF), mouse ES cells (ES), iPS cells generated by Oct4, Sox2, Klf4, c-Myc (OSKM), iPS cells generated with Combination #2+SKM (C2+SKM), iPS cells generated with Combination #3+SKM (C3+SKM), and iPS cells generated with Combination #4+SKM (C4+SKM), and MEFs transduced with lentivirus with an empty ORF+SKM (Empty+SKM). The samples marked early are MEFs transduced with the indicated factors before conversion into iPS cells (n=3 or 4). (B) Jensen-Shannon distance for conditions described in (A). (C) Number of differentially expressed genes for every pairwise comparison (p<0.05).

FIG. 12 demonstrates direct Oct4 targets. The direct targets of Oct4 from PluriNetWork (Som et al., 2010) were compared with ZFATF targets. Direct targets of Oct4 are colored light green. The direct targets of Oct4 or a ZFATF are colored purple. Protein-protein interactions are indicated by dashed lines. Activation of a gene is indicated by an arrow, while repression is indicated by a red segment ending with a perpendicular bar. The size of the node reflects level of expression in OSKM iPS cells.

FIGS. 13A-13B demonstrate that ATFs target key regulators of pluripotency. (A) Specificity and Energy Landscapes (SELs) of the five ATFs found in this study to replace Oct4. The first two ATFs (blue and orange) appear in all three combinations. The SEL displayed shows data after three rounds of enrichment. (B) Target genes of the ATFs in Combination #2 when the top 100 scoring 10-bp motifs are used from CSI. These genes have an ATF target site ±1 kb of the TSS and are upregulated >2-fold in cells with ATFs expressed. Colors indicate gene ontology: pluripotency (purple), proliferation (red), suppressor of differentiation (gray), morphogenesis (teal), and germline development (pink). The size of the node highlights their importance as upstream regulators.

FIG. 14 presents twelve exemplary ATFs and their respective DNA sequences.

FIGS. 15A-15C demonstrate transcriptional networks activated by ATFs and Oct4. (A) Nodes of the pluripotency network activated by the ATFs of C2. The size of the node reflects level of expression in C2+SKM iPS cells. (B) ChIP read density in C2+SKM early cells for HA-tagged ATFs for 17 direct target genes (left) and 15 indirect target genes (right) for nodes in FIG. 6A. Traces represent coverage across a window of −2 kb to +1.5 kb relative to the TSS. (C) Direct targets of Oct4. The size of the node reflects level of expression in Oct4+SKM iPS cells.

FIG. 16 illustrates a ligation-independent cloning strategy which relies on long overhangs to improve the efficiency of insertion of the cassette to the vector.

FIG. 17 illustrates a T4 DNA polymerase exonuclease reaction. A 30-bp overhang consists of three nucleotides until the position at which exonuclease activity is designed to be terminated.

FIGS. 18A-18C illustrates a particular embodiment of ATF-mediated cardiomyocyte differentiation by modulation of the Wnt pathway on H9 c-TnnT-pGZ cells. (A) ES cells were maintained in E8 until nearly confluent. On Day 0, the GSK3 inhibitor, CHIR99021 was added to the differentiation medium (RPMI 1640+B27 without insulin) to promote differentiation into mesoderm. On Day 3, the Wnt maturation inhibitor, IWP2 was added to promoted differentiation into cardiomyocytes. On Day 10, the expression of TNNT2 was evaluated by the expression of GFP. From Day 9 onwards, cardiomyocytes are maintained in RPMI 1640+B27 with insulin. (B) The ATF library was tested to replace the function of the Wnt pathway inhibitors. Human pluripotent stem cells bearing a reporter for TNNT2 were transduced with lentiviruses encoding either ATFs or no open reading frame (Empty). Cells were treated with puromycin to select for transduced cells. The differentiation protocol was performed in the presence or absence of one or more inhibitors. (C) ATFs were capable of replacing the Wnt maturation inhibitor, IWP2. Approximately 21% of the ATF library +CHIR-treated cells were GFP⁺. Only 1.1% of the Empty+CHIR-treated cells were GFP⁺, so the expected false positive rate is approximately 5.3%. Cells of the ATF library +CHIR formed larger sheaths of cardiomyocytes with regular beating compared to the Empty +CHIR treatment.

FIGS. 19A-19D demonstrate use of an ATF library to promote cardiomyocyte differentiation. (A) Genetic screen with an ATF library. ATFs are comprised of an interaction domain, 3-zinc fingers, nuclear localization signal, VP64 activation domain, and 3×HA tag. The library has a complexity of 2.6×10⁶ ATFs, each of which can target a 9-bp sequence. (B) A cardiomyocyte lineage-specific reporter cell line was created by lentiviral delivery of the human troponin T (TNNT2) promoter driving expression of copGFP and zeocin resistance (cTnnT-pGZ). (C) Flow cytometry results at Day 10 after treatment with CHIR99021. GFP⁺ H9 cTnnT-pGZ cells transduced with the ATF library were isolated as single cells for further analysis. Cells treated with lentivirus with an Empty ORF (negative control) were used to determine the frequency of false positive events. Percentages in the gated box are displayed in green. (D) ATFs were identified from 16 single cells by two-step nested PCR of genomic DNA. Unique ATFs are depicted with a different color. Two ATFs had a frameshift mutation shortly after the ID, coding for a protein that does not have a zinc finger structure. The frameshifted ATF and light blue ATF are expressed in 15/16 cells analyzed. Three ATFs are made up of two fingers (light blue, red, and purple). The combination, C3+CHIR, comprised of the frameshifted ATF (ATF5), blue ATF (ZFATF1), and orange ATF (ZFATF2), were retested in the validation step.

FIG. 20 demonstrates validation of ATFs from the cardiomyocyte screen. H9 cTnnT-pGZ cells were transduced with lentiviruses encoding ATFs. After selection for transduced cells, cells were treated with the GSK3 inhibitor, CHIR99021. On Day 3 of differentiation, the positive control was also treated with the Wnt maturation inhibitor, IWP2. No other condition was treated with IWP2. On Day 10, cells were evaluated for expression of GFP, structure of cardiomyocyte sheaths, and capacity to beat.

FIGS. 21A-21B present a representation of cardiomyocyte ATF sequences in the human genome. (A) The number of times the top 100 scoring CSI motifs appeared in the genome was quantified. Each data point represents a unique 10-bp sequence. (B) The number of times the top 100 scoring CSI motifs appeared within ±1kb of a TSS was quantified. Each data point represents a unique 10-bp sequence. The observed frequencies near the TSS are different from the expected frequencies (p<0.001 for df-2 and chi-squared=172).

FIGS. 22A-22C illustrate target genes of ATFs. The locations of the top 10 scoring 10-bp sequences from CSI were determined in the human genome. Genes highlighted in blue (early genes), green (middle genes), and yellow (late genes) have an ATF binding site within ±1kb of the TSS. (A) Target genes of ATF5, ZFATF1, and ZFATF2 (Treatment 10). (B) Target genes of ATF5 and ZFATF1 (Treatment 7). (C) Target genes of ATF5 and ZFATF2 (Treatment 9).

FIGS. 23A-23C illustrate use of the ATF library for hematopoietic differentiation. (A) Genetic screen with an ATF library. ATFs are comprised of an interaction domain, 3-zinc fingers, nuclear localization signal, VP64 activation domain, and 3×HA tag. The library has a complexity of 2.6×10⁶ ATFs, each of which can target a 9-bp sequence. (B) Inducing hematopoiesis. Human H1 ES cells are maintained in mTESR1™ medium (StemCell Technologies, Inc., Vancouver, CA) until the day of transduction. ATFs and natural factors are delivered by lentivirus on Day 0. From Day 1 onward, cells are maintained in 3F media, composed of mTeSR basal medium with FGF2, SCF, and thrombopoietin (3). Cells undergo 2 days of selection for transduction events with puromycin. On Day 10, cells are assessed for morphological changes to blood phenotypes. (C) ATF library+TAL1 generates cells of the hematopoietic lineage. Samples 1 and 2 are erythroid-like cells expanded on methylcellulose. Samples 3-5 are monocyte-like cells expanded on methylcellulose. Samples 2 and 4 were sequenced as cell clusters for ATF identification. Sample 2 was comprised of 4 ATFs; sample 4 was comprised of 15 ATFs.

FIG. 24 presents images validating ATFs from erythroid-like cluster. The parent cell type was human H1 ES cells. GATA2+ETV2 served as positive control for the myeloid lineage. TAL1+Empty control and TAL1+ATF5 (ATF with a frameshift mutation) induced differentiation but not to the same cell type as GATA2+ETV2. Differentiated cells after treatment with TAL1+ZFATF20 (middle image, last row) have a phenotype similar to myeloid cells. Cells treated with TAL1+ZFATF19 and TAL+ZFATF21 did not differentiate.

FIGS. 25A-25B demonstrate identification of ATF binding sites for hematopoiesis. (A) Cognate site identification (CSI). This method of determining the sequence specificity of DNA-binding factors involves incubating the ATF with randomized permutations of 25-bp sequences. The ATF-DNA complexes are captured with an antibody, and bound DNA is PCR-amplified for the next round of selection. Three rounds of selection are performed, and all three rounds are multiplexed and sequenced to obtain Specificity and Energy Landscapes (SELs) and position weight matrices (PWMs). SELs display the comprehensive binding preferences based on a chosen seed motif. The height of the peak is associated with affinity. Sequences that are 1-2 bp longer than the seed are arranged in concentric rings with respect to mismatches. Each ring outward from the 0-mismatch ring displays sequences to the corresponding number of mismatches. Within the mismatch rings, sequences are arranged by position of the mismatch, then alphabetically. (B) SELs of the four ATFs from the combination that induced differentiation into erythroid-like cells. Each SEL displayed shows data after three rounds of enrichment.

FIGS. 26A-26B represent erythroblast ATF sequences in the human genome. (A) The number of times the top 100 scoring CSI motifs appeared in the genome was quantified. Each data point represents a unique 10-bp sequence. (B) The number of times the top 100 scoring CSI motifs appeared within ±1 kb of a TSS was quantified. Each data point represents a unique 10-bp sequence. The observed frequencies near the TSS are different from the expected frequencies (p<0.001 for df-3 and chi-squared=14,195).

FIGS. 27A-27E illustrate use of the ATF library in HCMV gene expression. (A) Genetic screen with an ATF library. ATFs are comprised of an interaction domain, 3-zinc fingers, nuclear localization signal, VP64 activation domain, and 3×HA tag. The library has a complexity of 2.6×10⁶ ATFs, each of which can target a 9-bp sequence. (1) The ATF library was cloned into a second generation lentiviral vector. The screen is performed in cells with a robust change in phenotype or a lineage-specific reporter. (2) Positive outcomes are isolated as single cells, such that combinations of ATFs, if any, can be captured. (3) Integrated ATFs are identified from single cells. (4) Identified ATFs are retested for validation. Once validated, downstream experiments can be performed to identify ATF target genes. (B) Licensing the activation of HCMV genes. THP-1 monocytes were infected with HCMV (AD169) in which immediate-early gene 2 (IE2) fused to GFP is expressed when the virus enters lytic replication. (C) Flow cytometry results 20 hours post-infection with HCMV. THP-1 monocytes transduced with the ATF library or an Empty control were infected with HCMV encoding IE2-GFP fusion protein. Approximately 0.8% of cells treated with ATFs were GFP⁺ and 0.5% of the Empty control. THP-1 cells treated with valproic acid for 3 hours prior to HCMV infection served as the positive control (19% GFP⁺). Untreated THP-1 cells served as negative control (0% GFP⁺). ATF-treated cells with the greatest level of GFP expression (gate P4), were cell sorted. (D) ATFs were identified from 8 single cells by two-step nested PCR of genomic DNA. Unique ATFs are depicted with a different color. One ATF had a frameshift mutation shortly after the ID, coding for a protein that does not have a zinc finger structure. Three ATFs are made up of two fingers (light blue, pink, and red). (E) Validation of ATF hits by flow cytometry. The percentage of cells expressing IE2-GFP is displayed. Compared to the positive control (VPA), the ATFs do not activate HCMV gene expression in as many cells. The treatment numbers refer to the ATF combinations in (D). All treatments were infected with HCMV except for the first bar, uninfected.

FIGS. 28A-28B demonstrate identification of ATF binding sites for HCMV activation. (A) Cognate site identification (CSI). This method of determining the sequence specificity of DNA-binding factors involves incubating the ATF with randomized permutations of 25-bp sequences. The ATF-DNA complexes are captured with an antibody, and bound DNA is PCR-amplified for the next round of selection. Three rounds of selection are performed, and all three rounds are multiplexed and sequenced to obtain Specificity and Energy Landscapes (SELs) and position weight matrices (PWMs). SELs display the comprehensive binding preferences based on a chosen seed motif. The height of the peak is associated with affinity. Sequences that are 1-2 bp longer than the seed are arranged in concentric rings with respect to mismatches. Each ring outward from the 0-mismatch ring displays sequences to the corresponding number of mismatches. Within the mismatch rings, sequences are arranged by position of the mismatch, then alphabetically. (B) SELs of four ATFs that permit the expression of HCMV genes. Each SEL displayed shows data after three rounds of enrichment.

FIGS. 29A-29B represent HCMV ATF sequences in the human genome. (A) The number of times the top 100 scoring CSI motifs appeared in the genome was quantified. Each data point represents a unique 10-bp sequence. (B) The number of times the top 100 scoring CSI motifs appeared within +1kb of a TSS was quantified. Each data point represents a unique 10-bp sequence. The observed frequencies near the TSS are different from the expected frequencies (p<0.001 for df-3 and chi-squared=3,797).

While the present invention is susceptible to various modifications and alternative forms, exemplary embodiments thereof are shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description of exemplary embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though set forth in their entirety in the present application.

The methods and compositions provided herein are based at least in part on the Inventors' discovery of zinc finger-based artificial transcription factors (ATFs) that have the ability to promote cell fate conversions. These ATFs bind DNA through cooperative assembly and function as transcriptional activators. A combination of three ATFs facilitates the conversion of fibroblasts to induced pluripotent stem cells and the differentiation of stem cells to cardiomyocytes. The capacity of these ATFs to bind regulatory elements in the genome can enable various types of mammalian cell fate conversions, aiding in the generation of relevant cell types for cell therapies, drug screening, and disease modeling. Furthermore, the compositions and methods provided herein enable the targeting of gene-regulatory elements of mammalian systems and the resetting of the transcriptional circuitry.

Advantages of the claimed compositions and materials provided herein are at least three-fold. First, the ATFs of the present invention facilitate cell type conversions without a priori knowledge of potential key regulators, and can thereby reveal new gene networks and mechanistic pathways. The capacity of these ATFs to bind regulatory elements in the genome can enable various types of mammalian cell fate conversions, aiding in the generation of relevant cell types for cell therapies, drug screening, and disease modeling. Second, the three-zinc finger ATFs provided herein are capable of activating many fold over background. Some of these three-zinc finger ATFs activate expression 329-fold over background levels. Third, the short sequence bound by zinc fingers means that such DNA-binding elements are fairly ‘promiscuous’ in the genome, so it was not clear that an ATF based on this, where expression profiling was intended, would be effective or satisfactory.

Accordingly, in a first aspect, provided herein are engineered zinc finger proteins (“artificial transcription factors” or ATFs) capable of precise regulation of gene expression at a given locus. As used herein, the term “artificial transcription factor” refers to an engineered or “non-naturally occurring” zinc finger protein or fusion protein that binds to a nucleic acid (e.g., DNA, RNA) and/or protein. Encompassed by the term ATF are engineered zinc fingers comprising at least one zinc finger domain, typically two zinc finger domains, three zinc finger domains, or more. A zinc finger domain is a DNA binding motif of DNA binding proteins that are most frequently discovered in eukaryotes, which is discovered in various species from yeast to higher plant life and human beings. In the present disclosure, the zinc finger domain may be a Cys2-His2 type, where two, three, or more zinc finger domains are arranged in parallel to constitute a zing finger protein.

As used herein, the term “non-naturally occurring” means, for example, one or more of the following: (a) a polypeptide comprised of a non-naturally occurring amino acid sequence; (b) a polypeptide having a non-naturally occurring secondary structure not associated with the polypeptide as it occurs in nature; (c) a polypeptide that includes one or more amino acids not normally associated with the species of organism in which that polypeptide occurs in nature; (d) a polypeptide that includes a stereoisomer of one or more of the amino acids comprising the polypeptide, which stereoisomer is not associated with the polypeptide as it occurs in nature; (e) a polypeptide that includes one or more chemical moieties other than one of the natural amino acids; or (f) an isolated portion of a naturally occurring amino acid sequence (e.g., a truncated sequence).

In preferred embodiments, ATFs are designed to have a particular ATF formula including two or three zinc finger domains, a nuclear localization signal (NLS), and a specific interaction domain (ID). Twelve exemplary ATFs and their respective DNA sequence open reading frames are presented in FIG. 14.

In a preferred embodiment, provided herein are ATFs comprising a polydactyl zinc finger protein comprising two or three zinc finger domains, an interaction domain, a nuclear localization signal, and an activation domain. Each of the two or three zinc finger domains comprises a variable residue region, where the variable residue positions are encoded by nucleotide sequence of SEQ ID NO:2 (VNN-TCC-VNN-VNN-CTC-ACC-VNN), where each “VNN” is a codon encoding an amino acid selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V. Presented another way, the variable residue positions of the two or three zinc finger domains is encoded by amino acid sequence of SEQ ID NO:3 (X⁻¹-S-X₂-X₃-L-T-X₆), where X⁻¹ is an amino acid preferably selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V; X₂ is preferably selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V; X₃ is preferably selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V; and X₆ is preferably selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V. For a three zinc finger-containing ATF, the zinc fingers are encoded by the following sequences:

Finger1: (SEQ ID NO: 4) CCTTACGCTTGCCCAGTGGAGTCCTGTGATCGCCGCTTCTCCVNNTCCVN NVNNCTCACCVNNCACATCCGCATCCACACAGGCCAGAAGCCC Finger2: (SEQ ID NO: 5) TTCCAGTGCCGCATCTGCATGCGCAACTTCAGCVNNAGCVNNVNNCTCAC CVNNCACATCCGCACCCACACAGGCGAA Finger3: (SEQ ID NO: 6) AAGCCCTTCGCCTGCGACATCTGTGGAAGAAAGTTTGCCVNNAGCVNNVN NCGCAAGVNNCATACCAAGATCCACTTGCGG where each “VNN” (underlined) reflects a variable residue within each zinc finger. Each VNN is a codon encoding an amino acid selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V.

ATF Architecture:

In general, ATFs of the invention comprise a zinc finger backbone, an interaction domain (ID), and an effect domain, and can include an optional nuclear localization signal (NLS). Since the ID interacts with the first finger of EGR1, it is located at the N-terminus to be functional for dimerization with a second ATF. The other elements may be present in any order.

In some cases, the interaction domain (ID) comprises a 15-amino acid peptide that enables interaction with the hydrophobic face of the most 5′ zinc finger domain. In other cases, the ID comprises fewer than or greater than 15 amino acids. For example, the ID can comprise a peptide having 10-20 amino acids (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids). In some cases, the effector domain is an activation domain. In other cases, the effector domain is a repression domain. Suitable effector domains include, without limitation, VP64 (an activation domain) and KRAB (a repression domain). Other examples are set forth in Eguchi et al., 2014.

In some cases, the activation domain is a sequence derived from VP64. VP64 is a transcriptional activator composed of four tandem copies of VP16 (Herpes Simplex Viral Protein 16, amino acids 437-447, DALDDFDLDML; SEQ ID NO:7), generally connected with glycine-serine linkers. When fused to another protein domain that can bind near the promoter of a gene, VP64 acts as a strong transcriptional activator. Other activation domains appropriate for use according to the ATF's provided herein include, without limitation, the RelA activation domain (PGLPNGLLSGDEDFSSIADMDFSALLSQISS; SEQ ID NO:8), Beta-catenin (FDTDL; SEQ ID NO:9), amphipathic helix (PEFPGIELQELQELQALLQQ; SEQ ID NO:10), RNA-based activation domains (see, for example, Saha, S., Ansari, A. Z., Jarrell, K. A., Ptashne, M. and Jarell, K. A. (2003) RNA sequences that work as transcriptional activating regions. Nucleic Acids Res. 31, 1565-1570), and other non-protein based activation domains. For example, synthetic molecules that mimic transcription factors include wrenchnolol or a wrenchnolol derivative, isoxazoladine, and peptoids as reviewed in Eguchi et al., 2014. See also Jung et al. (J. Am. Chem. Soc. 2009, 131(13):4774-4782).

The ability of a domain to activate transcription can be validated by fusing the domain to a known DNA binding domain and then determining if a reporter gene operably linked to sites recognized by the known DNA-binding domain is activated by the fusion protein.

Nuclear localization signals (NLS) are amino acid motifs conferring nuclear import through binding to proteins defined by gene ontology GO:0008139, for example clusters of basic amino acids containing a lysine residue (K) followed by a lysine (K) or arginine residue (R), followed by any amino acid (X), followed by a lysine or arginine residue (K-K/R-X-K/R consensus sequence, Chelsky D. et al., 1989 Mol Cell Biol 9, 2487-2492). In preferred embodiments, the NLS can be derived from human EGR1 (early growth response 1). EGR1, which is also known as Zif268 (zinc finger protein 225) or NGFI-A (nerve growth factor-induced protein A), is a mammalian transcription factor that in humans is encoded by the EGR1 gene. In some cases, the EGR1 NLS sequence is KDKKADKSVV (SEQ ID NO: 11). In other cases, a NLS sequence suitable for use according to the invention provided herein is PKKKRKV (SEQ ID NO:12), which is the simian virus 40 large T-antigen (Kalderon et al. (1984) A short amino acid sequence able to specify nuclear location. Cell 39, 499-509).

In some cases, ATFs of the invention further comprise a dimerization domain. An alternative method of linking DNA binding domains is the use of dimerization domains, especially heterodimerization domains (see, e.g., Pomerantz et al (1998) Biochemistry 37: 965-970). In this implementation, DNA binding domains are present in separate polypeptide chains. For example, a first polypeptide encodes DNA binding domain A, linker, and domain B, while a second polypeptide encodes domain C, linker, and domain D. An artisan can select a dimerization domain from the many well-characterized dimerization domains. Domains that favor heterodimerization can be used if homodimers are not desired. A particularly adaptable dimerization domain is the coiled-coil motif, e.g., a dimeric parallel or anti-parallel coiled-coil. Coiled-coil sequences that preferentially form heterodimers are also available (Lumb and Kim, (1995) Biochemistry 34: 8642-8648). Another species of dimerization domain is one in which dimerization is triggered by a small molecule or by a signaling event. Such dimerization domains can be utilized to provide additional levels of regulation.

ATFs for Inducing Pluripotency:

In another aspect, provided herein are methods of using ATFs and combinations of ATFs useful to induce pluripotency in mammalian somatic cells. As described in the Examples that follow, combinations of ATFs have been shown to induce somatic cell reprogramming when provided to a somatic cell in combination with potency determining factors such as Sox2, Klf4, and c-Myc. For example, provided herein is a method of reprogramming a somatic cell to pluripotency, where the method comprises (a) exposing a somatic cell to a plurality of artificial transcription factors, wherein the artificial transcription factors are selected from the group consisting of ZFATF1, ZFATF2, ZFATF3, ZFATF4, and ZFATF5; (b) further exposing the somatic cell to a plurality of potency determining factors comprising Sox2, Klf4, and c-Myc; and (c) culturing the exposed cells to obtain reprogrammed cells having a higher potency level than the somatic cell. Combinations of ATFs useful for such methods include, without limitation, (1) ZFATF1, ZFATF2, and ZFATF3; (2) ZFATF1, ZFATF2, and ZFATF4; and (3) ZFATF1, ZFATF2, and ZFATF5. Other ATFs include ZFATF6, ZFATF7, ZFATF8, ZFATF9, ZFATF10, ZFATF11, and ZATF12.

Advantageously, the present invention allows the generation of pluripotent cells, such as iPS cells, from somatic cells without requiring an addition of cell surface receptors for introducing the potency-determining factors to the somatic cells. As used herein, the term “reprogramming” refers to a genetic process whereby differentiated somatic cells are converted into de-differentiated, pluripotent cells, and thus have a greater pluripotency potential than the cells from which they were derived. That is, the reprogrammed cells express at least one of the following pluripotent cell-specific markers: SSEA-1, SSEA-3, SSEA-4, TRA-1-60, or TRA 1-81. When the ATF library was tested for reprogramming of mouse fibroblasts, the resulting mouse pluripotent cells expressed SSEA-1. Other mouse markers include Oct4, Nanog, Esrrb, Utf1, Lin28a, and Dppa2 (Buganim Y, et al. (2012) Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase. Cell 150(6):1209-1222).

As used herein, “pluripotent cells” refer to a population of cells that can differentiate into all three germ layers (e.g., endoderm, mesoderm and ectoderm). Pluripotent cells express a variety of pluripotent cell-specific markers, have a cell morphology characteristic of undifferentiated cells (i.e., compact colony, high nucleus to cytoplasm ratio and prominent nucleolus) and form teratomas when introduced into an immunocompromised animal, such as a SCID mouse. The teratomas typically contain cells or tissues characteristic of all three germ layers. One of ordinary skill in the art can assess these characteristics by using techniques commonly used in the art. See, e.g., Thomson et al., Science 282:1145-1147 (1998). Pluripotent cells are capable of both proliferation in cell culture and differentiation towards a variety of lineage-restricted cell populations that exhibit multipotent properties. Multipotent somatic cells are more differentiated relative to pluripotent cells, but are not terminally differentiated. Pluripotent cells therefore have a higher potency than multipotent cells. As used herein, “reprogrammed pluripotent primate stem cells” (and similar references) refer to the pluripotent products of somatic cell reprogramming methods. Such cells are suitable for use in research and therapeutic applications currently envisioned for human ES cells.

As used herein, a “potency-determining factor” refers to a factor, such as a gene or other nucleic acid, or a functional fragment thereof, as well as an encoded factor or functional fragment thereof, used to increase the potency of a somatic cell, so that it becomes pluripotent. The potency-determining factors optionally can be present only transiently in the reprogrammed cells or can be maintained in a transcriptionally active or inactive state in the genome of the reprogrammed cells. Likewise, the potency-determining factors can be present in more than one copy in the reprogrammed cells, where the potency-determining factor can be integrated in the cell's genome, can be extra-chromosomal or both.

Generally, methods for identifying potency-determining factors include the steps of introducing genetic material encoding one or a plurality of putative potency-determining factors into somatic cells receptive to uptake of the genetic material under conditions effective to express the factors encoded on the introduced genetic material at levels sufficient to reprogram the cells to a less differentiated, higher-potency state; and observing a population of pluripotent cells after introduction of the genetic material. The pluripotent cells can be characterized by cell morphology, pluripotent cell-specific markers or both. Advantageously, the pluripotent cells can be identified by expression in the treated cells of a marker provided in the cells so as to be expressed only upon reprogramming of the cells to a pluripotent state. Through this approach, potency-determining factors capable of reprogramming somatic cells into pluripotent cells can be identified, as is described in the examples below.

Suitable somatic cells can be any somatic cell, although higher reprogramming frequencies are observed when the starting somatic cells have a doubling time about twenty-four hours. Somatic cells useful in the invention are non-embryonic cells obtained from a fetal, newborn, juvenile or adult primate, including a human. Examples of somatic cells that can be used with the methods described herein include, but are not limited to, bone marrow cells, epithelial cells, fibroblast cells, hematopoietic cells, hepatic cells, intestinal cells, mesenchymal cells, myeloid precursor cells and spleen cells. Alternatively, the somatic cells can be cells that can themselves proliferate and differentiate into other types of cells, including blood stem cells, muscle/bone stem cells, brain stem cells and liver stem cells. Multipotent hematopoietic cells, suitably myeloid precursor or mesenchymal cells, are specifically contemplated as suited for use in the methods of the invention.

In some cases, the methods comprises exposing or introducing into a somatic cell a genetic construct that enables efficient and robust delivery of ATFs and potency-determining factors to most cell types, including non-dividing and hard-to-transfect cells (primary, blood, stem cells) in vitro or in vivo. Viral-based constructs integrated into genomic DNA result in high expression levels. In addition to a DNA segment that encodes a potency-determining factor of interest, the vectors include a transcription promoter and a polyadenylation signal operatively linked, upstream and downstream, respectively, to the DNA segment. The vector can include a single DNA segment encoding a single potency-determining factor or a plurality of potency-determining factor-encoding DNA segments. A plurality of vectors can be introduced into a single somatic cell. The vector can optionally encode a selectable marker to identify cells that have taken up and express the vector. As an example, when the vector confers antibiotic resistance on the cells, antibiotic can be added to the culture medium to identify successful introduction of the vector into the cells. Integrating vectors can be employed, as in the examples, to demonstrate proof of concept. Retroviral (e.g., lentiviral) vectors are integrating vectors; however, non-integrating, episomal vectors can also be used. Such vectors can be lost from cells by dilution after reprogramming, as desired. A suitable episomal non-integrating vector is an Epstein-Barr virus (EBV) vector.

Alternative delivery methods for artificial transcription factors of the invention include transfer of nucleic acids by transfection or via viral vectors such as, but not limited to, herpes virus-, adeno virus- and adeno-associated virus-based vectors.

ATF Libraries:

In another aspect, provided herein is a library of ATFs designed to bind to and modulate the expression of a gene product. In some cases, the gene product can be a component of a cellular pathway (e.g., signaling pathway) associated with development of particular cell types. In other cases, the gene product can be a component of a cellular pathway associated with cell potency. As used herein, the term “library” is used according to its common usage in the art to denote a collection of polypeptides or, preferably, nucleic acids encoding polypeptides.

In some cases, ATFs provided herein contain regions of randomization, such that each library of ATFs will comprise or encode a repertoire of polypeptides, wherein individual polypeptides differ in sequence from each other. The same principle is present in virtually all libraries developed for selection, such as by phage display. As used herein, “randomization” refers to the variation of the sequence of the polypeptides which comprise the library, such that various amino acids may be present at any given position in different polypeptides. Randomization may be complete, such that any amino acid may be present at a given position, or partial, such that only certain amino acids are present. Preferably, the randomization is achieved by mutagenesis at the nucleic acid level, for example by synthesizing novel genes encoding mutant proteins and expressing these to obtain a variety of different proteins. Alternatively, existing genes can be themselves mutated, such by site-directed or random mutagenesis, in order to obtain the desired mutant genes.

In general, an ATF library can be obtained using any appropriate molecular biology techniques. As described in the Examples that follow, an exemplary method for obtaining a zinc finger ATF library comprises ligation-independent cloning. In particular, ligation-independent cloning into a second-generation lentiviral expression vector that contains the ccdB toxin was performed to eliminate background colonies. The first zinc finger was ordered as an oligomer with VNN codons in the variable residue positions (−1, 2, 3, and 6 of the alpha helix). The second and third zinc fingers were ordered as a separate oligo with VNN codons in the variable residue positions (−1, 2, 3, and 6 of the alpha helix). These two oligomers were amplified by PCR in a fusion reaction with a third oligomer containing the nuclear localization signal, VP64, and the HA tag. Amplified fragments were flanked by 30-bp regions with homology to the backbone vector. These homologous arms were treated with an exonuclease to create long overhangs, then cloned into the backbone vector. Plasmids comprising the oligomers cloned into the backbone vector can be transformed in highly competent bacteria.

In some cases, an ATF library can include additional variations that repress gene expression or modify chromatin.

In some cases, an ATF library as provided herein can be in cells such as engineered cells, or delivered to cells or tissues as RNA or full proteins to achieve the desired effects. This could be useful for research or therapeutic purposes.

Methods

In another aspect, provided herein are methods of using ATFs and ATF libraries as provided herein. In particular, ATFs can be used in methods for altering the potency of a cell and/or to promote differentiation of a cell into one or more particular cell types or cell lineages.

In some cases, the ATFs and ATF libraries provided herein can be used in combination with various genetic tools. For example, an ATF library can be screened for factors capable of inducing pluripotency in somatic cells (e.g., mouse embryonic fibroblasts) when expressed in the somatic cell in combination with three of the four recognized reprogramming factors (i.e., Sox2, Klf4, and c-Myc, but not Oct4). Genetic tools such as cognate site identification of the ATF binding sites, global transcriptional profiling, and characterization of the genome-wide epigenetic landscapes can be used to identify ATFs having a capacity to reprogram the somatic cell to a pluripotent state even in the absence of exogenous Oct4.

In some cases, an ATF library can be screened to identify ATFs capable of inducing differentiation of pluripotent cells such as human embryonic stem cells (ESCs) or human iPS cells into a differentiated cell type. For example, an ATF library can be screened using various combinations of Wnt pathway activators and inhibitors such as CHIR99021 (“CHIR”; a specific inhibitor of GSK3 for activation of canonical Wnt signaling) and IWP2 (which inhibits Wnt) to identify ATFs capable of standing in place of a Wnt inhibitor to induce cardiomyocyte differentiation in the presence of CHIR.

In certain embodiments, provided herein is a method of directing differentiation of a pluripotent stem cell to a cardiomyocyte. In some cases, the differentiation method comprises (a) exposing a pluripotent stem cell to one or more artificial transcription factors (ATFs) selected from the group consisting of ZFATF1, ZFATF2, and ATF5; and (b) culturing the exposed cells of (a) in the presence of an activator of Wnt/β-catenin signaling for about 7-10 days, such that a population of cells comprising cardiomyocytes is obtained. In some cases, the ATFs comprise ZFATF1, ZFATF2, and ATF5.

In some embodiments, activation of Wnt/β-catenin signaling is achieved by inhibiting Gsk3 phosphotransferase activity or Gsk3 binding interactions. Gsk3 inhibition can be achieved in a variety of ways including, but not limited to, providing small molecules that inhibit Gsk3 phosphotransferase activity, RNA interference knockdown of Gsk3, and overexpression of dominant negative form of Gsk3. Dominant negative forms of Gsk3 are known in the art as described, e.g., in Hagen et al. (2002), J. Biol. Chem., 277(26):23330-23335, which describes a Gsk3 comprising a R96A mutation.

In some embodiments, Gsk3 is inhibited by contacting a cell with a small molecule that inhibits Gsk3 phosphotransferase activity or Gsk3 binding interactions. Suitable small molecule Gsk3 inhibitors include, but are not limited to, CHIR99021 (“CHIR”), CHIR98014, BIO-acetoxime, BIO, LiCl, SB 216763, SB 415286, AR A014418, 1-Azakenpaullone, Bis-7-indolylmaleimide, and any combinations thereof. In some embodiments, any of CHIR99021, CHIR98014, and BIO-acetoxime are used to inhibit Gsk3 in pluripotent stem cells in the differentiation methods described herein. In one embodiment, the small molecule Gsk3 inhibitor to be used is CHIR99021 at a concentration ranging from about 3 μM to about 12 μM, e.g., about 3 μM, 4 μM, 5 M, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM or another concentration of CHIR99021 from about 3 μM to about 12 μM. In another embodiment, the small molecule Gsk3 inhibitor to be used is CHIR98014 at a concentration ranging from about 0.1 μM to about 1 μM, e.g., about 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM or another concentration of CHIR98014 from about 0.1 μM to about 1 μM. In another embodiment, the small molecule Gsk3 inhibitor to be used is BIO-acetoxime at a concentration ranging from about 0.1 μM to about 1 μM, e.g., about 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM or another concentration of BIO-acetoxime from about 0.1 μM to about 1 μM.

In other embodiments, Gsk3 activity is inhibited by RNA interference knockdown of Gsk3. For example, Gsk3 expression levels can be knocked-down using commercially available siRNAs against Gsk3, e.g., SignalSilence® GSK-3α/β siRNA (catalog #6301 from Cell Signaling Technology®, Danvers, Mass.), or a retroviral vector with an inducible expression cassette for Gsk3, e.g., a commercially available Tet-inducible retroviral RNA interference (RNAi) system from Clontech (Mountain View, Calif., Catalog No. 630926), or a cumate-inducible system from Systems Biosciences, Inc. (Mountain View, Calif.), e.g., the SparQ® system, catalog no. QM200PA-2.

In another aspect, provided herein is a method for directing differentiation of a pluripotent stem cell to a hematopoietic lineage. In some cases, the method comprises (a) exposing a pluripotent stem cell to two or more artificial transcription factors (ATFs) selected from the group consisting of ZFATF19, ZFATF20, ZFATF21, ATF5, ZFATF1, ZFATF6, ZFATF10, ZFATF13, ZFATF17, ZFATF22, ZFATF23, ZFATF24, ZFATF25, ZFATF26, ZFATF27, ZFATF28, ZFATF29, ZFATF30, and ZFATF31; and (b) culturing the exposed cells of (a) in a basal culture medium comprising FGF2, SCF, and thrombopoietin, and in the presence of TAL1 for about 7-10 days, such that a cell population comprising hematopoietic lineage cells is obtained. In some cases, the ATFs comprise ZFATF19, ZFATF20, ZFATF21, and ATF5, and the cell population comprises erythroid-like cells. In other cases, the ATFs comprise ZFATF1, ZFATF6, ZFATF10, ZFATF13, ZFATF17, ZFATF22, ZFATF23, ZFATF24, ZFATF25, ZFATF26, ZFATF27, ZFATF28, ZFATF29, ZFATF30, and ZFATF31, and the cell population comprises monocyte-like cells.

In other cases, an ATF library can be screened to identify ATFs capable of inducing differentiation of pluripotent and/or multipotent cells to various blood lineages. For example, an ATF library can be screened for ATFs capable of differentiating human pluripotent stem cells (e.g., iPS cells, embryonic stem cells) into erythroblast-like cells and monoblast-like cells. As described in the Examples that follow, screening an ATF library identified factors that promoted the differentiation of ESCs into blood lineages, and found combinations of ATFs (with TAL1(SCL)) that produced a range of phenotypes from monoblast-like to erythroblast-like cells.

In some cases, the methods provided herein include screening an ATF library for factors that promote the reactivation of latent cytomegalovirus (CMV) virus infections, the inventors identified eight ATF combinations, which can help inform the field's understanding of how CMV and its reactivation from a latent state to lytic replication is regulated.

Articles of Manufacture

In another aspect, provided herein is an article of manufacture such as a kit comprising a plurality of artificial transcription factors or an ATF library as described herein. In certain embodiments, the kit comprises (i) a plurality of ATFs selected from the group consisting of ZFATF1, ZFATF2, ZFATF3, ZFATF4, and ZFATF5; (ii) a plurality of potency determining factors such as Sox2, Klf4, and c-Myc; and (iii) instructions describing a method for reprogramming a somatic cell to pluripotency, the method employing the ATFs and potency determining factors. In some cases, a kit provided herein further comprises or alternatively comprises instructions for designing artificial transcription factors.

It is understood that certain adaptations of the invention described in this disclosure are a matter of routine optimization for those skilled in the art, and can be implemented without departing from the spirit of the invention, or the scope of the appended claims. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. It is understood, however, that examples and embodiments of the present invention set forth above are illustrative and not intended to confine the invention. The invention embraces all modified forms of the examples and embodiments as come within the scope of the following claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described herein.

In the specification and in the claims, the terms “including” and “comprising” are open-ended terms and should be interpreted to mean “including, but not limited to . . . .” These terms encompass the more restrictive terms “consisting essentially of” and “consisting of.” As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. As well, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising,” “including,” “characterized by,” and “having” can be used interchangeably.

As used herein, “about” means within 5% of a stated concentration, concentration range, or amount, or within 5% of a stated time frame.

As used herein, “effective amount” means an amount of an agent sufficient to evoke a specified cellular effect according to the present invention.

The invention will be more fully understood upon consideration of the following non-limiting Examples.

EXAMPLES Example 1: Artificial Transcription Factor Library-Guided Cell Fate

In this Example, we demonstrate that Artificial Transcription Factors (ATFs), which are tailor-made molecules designed to bind DNA and regulate transcription in a desired manner, take cells through a different path to pluripotency compared to cells expressing exogenous Oct4.

Methods

Zinc Finger ATF Constructs for Testing Architecture:

The first two or all three zinc fingers of human EGR1 were used to test the ATF architecture. The peptide sequence (HPMNNLLNYVVPKMR (SEQ ID NO: 13)) preceding the DNA-binding domain served as the interaction domain. The native nuclear localization signal for EGR1 was used to ensure entry into the nucleus. A tetrameric repeat of VP16 (VP64) served as the activation domain. Three repeats of hemagglutinin (HA) were used as a C-terminal epitope tag.

Zinc Finger ATF Library:

The scaffold of the ATF is comprised from N- to C-terminus: a 15-amino acid interaction domain, the DNA binding domain of human EGR1, NLS from EGR1, VP64, and 3×HA tag. The ATF library was created by amplifying oligos with VNN codons at the −1, 2, 3, and 6 positions relative to the recognition helix of each zinc finger. The ATF library was cloned into the second-generation pSIN vector by ligation-independent cloning. Estimation of the library complexity was measured by plating a fraction of the transformants on plates, and counting the number of colonies that grew from the fraction.

The zinc finger ATF library was created by ligation-independent cloning into a second-generation lentiviral expression vector that contains the ccdB toxin to eliminate background colonies (FIG. 16). Ligation-independent cloning relies on long overhangs that improve the efficiency of insertion of the cassette to the vector. 30-bp overhangs were created by treating with T4 DNA polymerase with 3′ to 5′ exonuclease activity (FIG. 17). The nucleotide content in the overhangs are such that a specific nucleotide is missing. The missing nucleotide was added to the reaction to terminate exonuclease activity in a predetermined manner. The chew-back of the ATF cassette was terminated with inclusion of cytosine and the pSIN vector with guanine. Homologous 30-bp overhangs were ligated together in a highly reliable manner without a DNA ligase. The long overhangs also increase the efficiency of ligation of the cassette to the vector.

A cassette containing the ccdB gene was cloned into pSIN with EcoRI and SpeI. The ccdB gene is encoded in the antisense direction

(SEQ ID NO: 14) 5′-GAATTCGGACCATACTCTATAATACTATCACTAACTTAATTAATTAT ATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTG GCTGAGATCAGCCACTTCTTCCCCGATAACGGAGACCGGCACACTGGCCA TATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGG TAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGG GATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCA CAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGC ATTTCACCAGCCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAA ACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCG GCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGA GATATTGACATCATATATGCCTTGAGCAACTGATAGCTGTCGCTGTCAAC TGTCACTGTAATACGCTGCTTCATAGCATACCTCTTTTTGACATACTTCG GGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAAT ACGCATACTGTTATCTGGCTTTTAGTAAGCCGGATCCACGCGGCGTTTAC GCCCCCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTG CCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGG CATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGG GGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAA CTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTT AGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATA TGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAA AACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATC CCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAAGTTCGGATGAG CATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGC TTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGT CTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTT TACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTC TCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATAC GCCCGGTAGTGATCTCGAGCCGGAAGCATAAAGTGTAAATTAATTAAGAT TAGAGTTAAGTAAGGTATTAGTGACACTAGT -3′.

The zinc finger ATF library was created by ordering the following oligos from IDT and fusing them to the ATF scaffold that includes the interaction domain, nuclear localization signal, activation domain, and 3×HA tag:

Oligo 1: (SEQ ID NO: 15) 5′-GGTACCATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGAA GATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCGCCGCTTCTCCV NNTCCVNNVNNCTCACCVNNCACATCCGCATCCACACAGGCCAGAAGCCC TTCCAGTGCCGCATCTGCATGCGCAACTTCAG -3′. Oligo 2: (SEQ ID NO: 16) 5′-CACAACACTTTTGTCTGCTTTCTTGTCCTTCTGCCGCAAGTGGATCT TGGTATGNNBCTTGCGNNBNNBGCTNNBGGCAAACTTTCTTCCACAGATG TCGCAGGCGAAGGGCTTTTCGCCTGTGTGGGTGCGGATGTGNNBGGTGAG NNBNNBGCTNNBGCTGAAGTTGCGCATGCAGATGC -3′.

where V stands for A, C, or G and B stands for C, G, or T

Fragment 3 (in pcDNA vector): (SEQ ID NO: 17) 5′-AAGGACAAGAAAGCAGACAAAAGTGTTGTGGGGCGCGCCGACGCGCT GGACGATTTCGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGATCTGGACATG CTCGGCTCCGATGCTCTGGACGATTTCGATCTCGATATGTTAATTAACTA CCCGTACGACGTTCCGGACTACGCTGGTTATCCCTATGACGTCCCGGATT ATGCAGGATCCTATCCATATGACGTTCCAGATTACGCTTGA-3′.

In the first PCR Oligo 2 and Fragment 3 are amplified together with forward primer 1 and reverse primer 1.

forward primer 1: (SEQ ID NO: 18) 5′- CCGCATCTGCATGCGCAACTTCAGC -3′. reverse primer 1: (SEQ ID NO: 19) 5′- GCGGGCCCTCTAGACTCG -3′.

In the second PCR the product from the first PCR and Oligo1 are amplified together with forward primer 2 and reverse primer 2.

forward primer 2: (SEQ ID NO: 20) 5′-ACCATACTCTATAATACTATCACTAACTTAGCCGCCACCATGCATCC TATGAACAACCTGCTGAACTACGTGG -3′. reverse primer 2: (SEQ ID NO: 21) 5′-TCACTAATACCTTACTTAACTCTAATCTTAGTCAAGCGTAATCTGGA ACGTCATATGGATAGGATCCTGC -3′.

Both the ATF cassette and the pSIN-ccdB vector were digested with PacI and treated with T4 DNA polymerase, which has 3′ to 5′ exonuclease activity. The resulting vector was gel-extracted. The ligation was performed at a ratio of 1:6 (vector:cassette) without a DNA ligase. The ligation reaction was chemically transformed in ultracompetent XL-10 Gold cells. After 1 hour recovery in SOC media without antibiotic, the transformed culture was diluted in 150 mL SOC media with ampicillin (100 μg/mL). This culture was grown on a shaker at 37° C. for 12 hours. These cultures were maxiprepped for endotoxin-free purification of pSIN-plasmid. A small fraction ( 1/10,000) of the culture was plated on 4 plates before overnight incubation for counting purposes.

Estimation of the library complexity was measured by plating a fraction of the transformants on plates, and counting the number of colonies that grew from the fraction. A total of 210 transformations and 25 maxipreps were performed to cover the desired complexity.

The permutation of all 10-bp sequences is 4¹⁰=1,048,576. However, on duplex DNA, the complementary sequence will also be represented on the antisense strand. Palindromic sequences do not yield new sequences on the antisense strand. Therefore, N=(n−1)+(4^(n)+4^(n/2))/2, where N is the number of permutations on duplex DNA and n is the length in bp (if even) (2). For 10-bp sequences, there are 524,809 permutations that encompass the entire binding space on duplex DNA.

Sequencing of 100 clones revealed 60% of the ATFs contain functional ATFs (53% with 3 zinc fingers, 6% with 2 zinc fingers, and 1% with 4 zinc fingers). The remainder of sequences yielded poor reads (4%) or contained a frameshift mutation (36%). Accounting for the efficiency of creating functional ATFs, we predicted 4.37×10⁶ clones would be sufficient would be sufficient to achieve the desired complexity. Cloning of the library was repeated until the maxiprep cultures yielded a clone count of 5.56×10⁶ CFU.

To determine the diversity of the library, the sequences of 100 functional ATFs were determined by Sanger sequencing. All 16 possible amino acids encoded by VNN codons were represented in the recognition residues. This sample size is representative of the library at a 95% confidence interval and a margin of error of 9.8%.

Cell Culture:

Oct4:Cre^(mER-Cre-mER); mTmG mouse embryonic fibroblasts (MEFs) were grown in DMEM supplemented with 10% fetal bovine serum on plates coated with 0.1% gelatin. Mouse E14T ES cells and iPS cells were grown in knock-out DMEM supplemented with 15% fetal bovine serum, 1% nonessential amino acids, 2 mM L-glutamine, 1×10³ units/mL leukemia inhibitory factor, 1 mM sodium pyruvate, and 100 μM beta-mercaptoethanol. 4-hydroxytamoxifen was added at 100 nM concentration. Pluripotent cells were maintained on irradiated MEFs. Cells were maintained in a humidified 37° C. incubator with 5% CO₂.

Luciferase Assay:

The palindromic EGR1 binding site 5′-GCG-TGG-GCG-CGC-CCC-CGC-3′ (SEQ ID NO: 1) was cloned upstream of the luciferase gene in the pGL3 basic vector (Promega). HEK293 cells were transiently co-transfected with ATF in pcDNA-CMV, the pGL3 basic luciferase reporter, and a RSV-β-galactosidase reporter. The transfection was performed with Lipofectamine® 2000 (Thermo Fisher Scientific #11668019). Cells were collected and lysed 24 hours post-transfection. Luciferase assay (Promega #E4030) was performed according to manufacture guidelines, and readings were normalized to β-galactosidase levels to account for transfection efficiency. Significance values were obtained by one-way ANOVA of the log₁₀ transformed β-galactosidase-normalized luciferase values in SPSS Statistics 23.0.0.0 (IBM). A Tukey test was performed for post hoc analysis of treatments with statistically significant differences.

Retrovirus Production:

Oct4, Sox2, Klf4, and c-Myc were packaged into retrovirus with Plat-E cells as described in (Takahashi et al., 2007a).

Lentivirus Production:

ATFs and the empty control were packaged into lentivirus with HEK293FT cells using calcium phosphate transfection of pSIN expression, psPAX2 packaging, and pMD2.G envelope plasmids. Media containing virus was harvested 48-60 hours post-transfection. Lentivirus was centrifuged with a sucrose cushion at 25,000 rpm for 2 hours. Viral particles were suspended in PBS and concentrated virus was stored at −80° C. Viral titers were measured by counting cells that survived selection after 2 days in media containing puromycin (3 μg/mL). The ATF is driven by a constitutive promoter, EF1α.

Reprogramming to Pluripotent Cells:

Oct4: Cre^(mER-Cre-mER); mTmG MEFs were maintained on 0.1% gelatin in DMEM supplemented with 10% FBS until the day of transduction. Lentiviruses and retroviruses were delivered to MEFs with polybrene (8 μg/mL). The ATF Library was delivered at MOI=3, and validation of ATFs were performed with MOI=3 for each ATF. The Empty lentiviral control was delivered at an MOI=9. Oct4 (500 μL), Sox2 (500 μL), Klf4 (500 μL), and c-Myc (300 μL) were delivered to the cells at the same time of ATF transduction as fresh retrovirus. Lentivirus and retrovirus was removed 18 hours after transduction. Selection of lentiviral integration events was performed 42 hours post-transduction by addition of puromycin (3 μg/mL) for 2 days in mouse ES cell media (knock-out DMEM supplemented with 15% fetal bovine serum, 1% nonessential amino acids, 2 mM L-glutamine, 1×10³ units/mL leukemia inhibitory factor, 1 mM sodium pyruvate, and 100 μM beta-mercaptoethanol). Cells undergoing reprogramming were maintained in mouse ES cell media with 4-hydroxytamoxifen (100 nM) on irradiated MEFs. Fully reprogrammed colonies were picked and expanded. To obtain doubling times, iPS cells were grown on 0.1% gelatin.

Identification of ATFs from Single Cells:

Cells with a positive phenotype for Oct4 lineage tracing activation were isolated as single cells into a 96-well plate. Amplified regions were cloned into pcDNA with a CMV promoter. ATF sequences were deciphered by Sanger sequencing with a primer for the 3′ end of the CMV promoter.

TABLE 1 Primers for 2-Step Nested PCR Primer Sequence (5′ to 3′) ATF- TACTATCACTAACTTAGCCGCCACCATG forward-1 (SEQ ID NO: 23) ATF- GAGGGCA TCAGAACCCAGCATG reverse-1 (SEQ ID NO: 24) ATF- CATCAGCTAGCGCTGAACTACGTGGTGCCGAAG forward-2 (SEQ ID NO: 25) ATF- CGCGTCGGCGCGCCCCACAACACTTTTGTCTGC reverse-2 (SEQ ID NO: 26) CMV- CGCAAATGGGCGGTAGGCGTG forward (SEQ ID NO: 27)

The number of sequences obtained to reach saturation was determined by calculating the probability that the chance of finding another unique ATF is less than 1%: (1−1/(n+1))^(x)<0.01, where n is the number of unique ATFs identified and x is the number of sequences needed.

TABLE 2 Number of Sequences Necessary for Saturation # of Unique ATFs # of Sequences Identified (n) Needed (x) 3 17 4 21 5 26 6 30 7 35 8 40 9 44 10 49

Embryoid Body Formation:

For embryoid body (EB) formation, pluripotent cells were seeded into ultra-low adhesion dishes at a concentration of 1×10⁵ cells/mL in knock-out DMEM supplemented with 15% fetal bovine serum, 1% nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 μM beta-mercaptoethanol. Media was changed the day after seeding and every two days thereafter. EBs were collected on Day 5, 7, and 14 for RT-qPCR and immunofluorescence. Cells were maintained in a humidified 37° C. incubator with 5% CO₂.

Immunofluorescence:

EBs were plated on adherent plates on Day 5, 7, and 14 to culture EB outgrowths. iPS cells, EBs, or EB outgrowths were plated on glass slides coated with 0.1% gelatin for immunofluorescence. Antibody sources and dilutions are described in the Supplemental Experimental Procedures.

RT-qPCR:

RNA was extracted from cells with RNeasy Mini Kit (Qiagen #74104). RNA was converted into cDNA with SuperScript III First-Strand Synthesis System (Thermo Fisher #18080051). qPCR was performed with Bullseye EvaGreen qPCR Mix with low ROX (Midwest Scientific #BEQPCR-LR). Primer sets are listed in Table 3.

TABLE 3 Primers for qPCR SEQ  ID Primer Sequence (5′ to 3′) NO: Endogenous Oct4 TCAGTGATGCTGTTGATCAGG 28 forward Endogenous Oct4 GCTATCTACTGTGTGTCCCAGTC 29 reverse Endogenous Sox2 CCGTTTTCGTGGTCTTGTTT 30 forward Endogenous Sox2 TCAACCTGCATGGACATTTT 31 reverse Nanog forward AAACCAGTGGTTGAAGACTAGCAA 32 Nanog reverse GGTGCTGAGCCCTTCTGAATC 33 Lin28 forward GAAGAACATGCAGAAGCGAAGA 34 Lin28 reverse CCGCAGTTGTAGCACCTGTCT 35 Esrrb forward CACCTGCTAAAAAGCCATTGACT 36 Esrrb reverse CAACCCCTAGTAGATTCGAGACGAT 37 Utf1 forward GTCCCTCTCCGCGTTAGC 38 Utf1 reverse GGCAGGTTCGTCATTTTCC 39 T forward CTGTGGCTGCGCTTCAAGG 40 T reverse ATTGGGGTCCAGGCCTGAC 41 Nkx 2.5 forward TGACCCTGACCCAGCCAAAG 42 Nkx 2.5 reverse GAGAAGAGCACGCGTGGCTTC 43 Kdr forward CATCCCCCCAAGCTCAGCAC 44 Kdr reverse ACGCTGAGCATTGGGCCAAAG 45 Afp forward CACTGTCCAAGCAAAGCTGCG 46 Afp reverse GAGGTCAGCTGAGGGGCTTTC 47 Ttr forward GGATCCTGGGAGCCCTTTGC 48 Ttr reverse TCATCTGTGGTGAGCCCGTG 49 FoxA2 forward TGGGAGCCGTGAAGATGGAAG 50 FoxA2 reverse CAGGCCGGCGTTCATGTTG 51 Nes forward GCAGAGAGGCGCTGGAACAG 52 Nes reverse CCCTGCTTCTCCTGCTCCAG 53 Nef1 forward CTGGAGAAGCAGCTGCAGGAG 54 Nef1 reverse CTGGCCATCTCGCTCTTCGTG 55 Sox17 forward GCGGTTGGCACAGCAGAAC 56 Sox17 reverse GCTCGGCCTCTTCCACGAAG 57 GAPDH forward ACATCATCCCTGCATCCACT 58 GAPDH reverse CCTGCTTCACCACCTTCTTG 59

Chromatin Immunoprecipitation:

For chromatin immunoprecipitation, 5×10⁶ cells were fixed in 1.5% formaldehyde for 15 min. Harvested cells were flash frozen, then sonicated and lysed. Lysates were precleared and immunoprecipitated overnight with H3K27ac antibody (Abcam #ab4729), H3K9me3 antibody (Abcam #ab8898), or HA antibody (Abcam #ab9110) at 4° C. Immunoprecipitated histone marks were purified with protein G magnetic beads (Life Technologies #10004D) and a series of five washes. Cross-links of protein-DNA complexes were reversed by incubating at 65° C. for 6 hours. Eluted DNA was treated with RNase A and Proteinase K.

For histone marks, 6×10⁶ cells were harvested, and for ATFs, 2.5×10⁷ cells were harvested. Cells were fixed in 1.5% formaldehyde, then flash frozen. Cells were lysed with a series of 3 buffers: LB1, LB2, and LB3. LB1 consisted of 10 mM HEPES, 10 mM EDTA, 0.5 mM EGTA, and 0.25% Triton X-100. LB2 consisted of 200 mM NaCl, 10 mM HEPES, 1 mM EDTA, and 0.5 mM EGTA. LB3 consisted of 50 mM Tris-HCl, 10 mM EDTA, 0.5% Empigen BB, and 1% SDS. Samples were sonicated in a Misonix sonicator (S-4000) at 60% power, 10 sec on and 10 off, for a total of 32 min total pulse time. Samples were cleared by centrifugation 17,000×g for 10 min. For pre-clearing, samples were incubated with magnetic Protein G beads (Life Technologies #10003D) for 1 hour at 4° C. After pre-clearing, 1% of each sample was saved as input. Samples were incubated with the appropriate antibody, H3K27ac antibody (Abcam #ab4729), H3K9me3 antibody (Abcam #ab8898), or HA antibody (Abcam #ab9110), overnight at 4° C. with IP Buffer (2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, and 1% Triton X-100). Protein-DNA complexes on magnetic beads were washed in WB1 once, WB2, once, WB3 once, and TE Buffer twice. WB1 consisted of 2 mM EDTA, 20 mM Tris-HCl, 0.1% SDS, 1% Triton X-100, 150 mM NaCl). WB2 consisted of 2 mM EDTA, 20 mM Tris-HCl, 0.1% SDS, 1% Triton X-100, and 500 mM NaCl. WB3 consisted of 1 mM EDTA, 10 mM Tris-HCl, 250 mM LiCl, 1% deoxycholate, and 1% NP-40 detergent. DNA was eluted in 0.1 M NaHCO₃, 0.2 M NaCl, and 1% SDS (sodium dodecyl sulfate).

Protein-DNA complexes were reverse crosslinked by incubation at 65° C. for 6 hours. DNA was treated with RNase A and Proteinase K. Captured DNA was column purified (Epoch Life Sciences #1920-250). Samples were prepared for sequencing with the TruSeq ChIP Sample Preparation Kit (Illumina #IP-202-1012) as per the manufacturer instructions and quantified with a Qubit fluorometer (Life Technologies #Q32866). Three, four or six TruSeq indexed ChIP samples were pooled per lane. All samples were loaded at a final concentration of 8 pM and sequenced as single reads on the Illumina HiSeq 2500.

RNA-Seq Analysis:

Reads were aligned with Bowtie2 version 2.2.5 to either the human genome hg19 (HEK293) or mouse genome mm10 (MEFs or cells derived from MEFs). Counts were quantified with Cufflinks software, and differential expression was determined using the Cuffdiff program.

ChIP-Seq Analysis:

Reads were annotated to the mouse genome mm10 with Bowtie2 version 2.2.5. Output Sequence Alignment/Map (SAM) files of sequence data were converted to BAM files, which store the same data in a compressed, indexed, binary form, and then were sorted and indexed with Samtools 1.3. H3K27ac peaks were called with SPP and IDR using default settings. H3K27ac and H3K9me3 peaks were called with MACS2. Differential peak signals were determined by DiffBind 1.16.2. Spearman correlations were calculated with deepTools 2.2.2. ChIP peaks were visualized with Integrative Genomics Viewer (IGV). Coverage tracks were used to generate heatmaps and ChIP-seq profiles for multiple genes in deepTools.

Cognate Site Identification:

Cognate Site Identification was performed by incubating cell lysates containing zinc finger ATFs with a randomized library of 25-bp sequences. HEK293 cells were transiently transfected with ATFs using lipofectamine 2000 (Thermo Fisher #11668019). Cells that underwent a mock transfection (without an expression plasmid for ATFs) served as a negative control. Cells were harvested 48 hours post-transfection. Lysates were prepared by lysing 10⁷ cells in 300 μL of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, and 0.1% Na deoxycholate). A 21.5 G syringe needle was used for mechanical lysis. Lysates were centrifuged at 10 k×g for 10 min to separate precipitated genomic DNA from protein constituents. The supernatant, containing ATF protein, was used for CSI. HA magnetic beads (MBL #M132-9) were added to the cell lysates for immunoprecipitation. The binding reaction between the ATFs and 100 nM synthetic DNA was performed in binding buffer (25 mM HEPES, 80 mM KCl, 0.2 mM EDTA, 1 mM MgCl₂, 0.1 mM ZnSO₄) at 25° C. After pull-down, three quick washes with 100 μL ice-cold binding buffer were performed to remove unbound DNA. Magnetic beads were resuspended in a PCR master mix (Lucigen #30035-1) and the DNA was amplified for 15 or 18 cycles. Amplified DNA was column purified (Qiagen #28106), and this enriched DNA pool was used for the subsequent round of enrichment, for a total of three rounds. After three rounds of selection, Illumina sequencing adapters and a unique 6 bp barcode for multiplexing were added by PCR amplification. The starting library (Round 0) was also barcoded. Up to 180 samples were combined and sequenced in a single HiSeq2000 lane.

Cognate Site Identification Data Analysis:

Illumina sequencing yielded ˜180 million reads per lane. Reads were de-multiplexed by requiring an exact match to the 6 bp barcode and truncated to include only the 25 bp derived from the random portion of the library. On average, we obtained 709,300 reads per barcode. The occurrence of every k-mer (lengths 8 through 16 bp) was counted using a sliding window of size k. To correct for biases in our starting DNA library, we took the ratio of the counts of every k-mer to the expected number of counts in the mock-transfected control. The mock control was modeled using a 5^(th)-order Markov Model derived from the sequencing reads corresponding to the starting library (Round 0). We then calculated a

Z-score=(x−μ)/σ

or (CSI score minus mean)/standard deviation for each k-mer, using the distribution of k-mer enrichment values (CSI score) for the ATF.

Bioinformatic Analysis of ATF Binding Sites:

The genomic locations of the top five or 100 high-scoring 10-bp motifs from CSI were identified in the mm10 genome by the findMotif utility from the UCSC Genome Browser. These genomic sites were annotated using Homer. Annotated sites were filtered to those within +1 kb of the TSS. For correlation to RNA-seq data, only the genes exhibiting a 2-fold upregulation or more and found significant at p<0.05 in three pairwise comparisons (C2+SKM iPS vs. Empty+SKM early; C2+SKM early vs. Empty+SKM early; C2+SKM iPS vs. Oct4+SKM iPS) were included. For the other ATF-derived iPS lines, the condition C2+SKM was replaced with C3+SKM or C4+SKM. The identified ATF target genes were included as nodes in the transcriptional network if they were previously implicated in pluripotency (Buganim et al., Cell 150(6):1209-1222, 2012; Heng et al., Cell Stem Cell 6(2):167-174, 2010; Kim et al., Cell 132(6):1049-1061, 2008; Krentz et al., Dev Biol 377(1):67-78, 2013; Lujan et al., Nature 521(7552):352-356, 2015; Marson et al., Cell 134(3):521-533, 2008; Sharov et al., BMC Genomics 9(1):269, 2008; Shu et al., Cell 153(5):963-975, 2013; Som et al., PLoS ONE 5(12):e15165, 2010). The STRING database was used to identify protein-protein interactions.

Results

ATF Architecture and Library Design:

To determine the best architecture for a zinc finger ATF library, we tested the impact of each modular domain on the level of induction. The zinc finger backbone is derived from human Early Growth Response 1 (EGR1) (also known as Transcription Factor ZIF268), a well-studied scaffold for zinc finger ATFs. EGR1 comprises C2H2-type zinc fingers which bind to DNA motifs having the sequence 5′-GCG(T/G)GGGCG-3′. We fused VP64, a tetrameric repeat of the 11-amino acid activation region of VP16, a potent transactivation domain from the herpes simplex virus (FIG. 1A) (Triezenberg et al., 1988). To the N-terminus of the zinc fingers, we attached a 15-amino acid peptide that serves as an interaction domain (ID), reported to allow dimerization of the ATF to another ATF with the hydrophobic surface of the first zinc finger of EGR1 (Wang et al., 2001). This ID adds a novel layer of control to the ATF library by allowing the ATFs to capitalize on cooperative binding (Moretti and Ansari, 2008). The nuclear localization signal (NLS) comes from the native NLS of EGR1. A palindromic cognate site for EGR1 was placed upstream of a luciferase reporter to test the contribution of each domain in activating the reporter. The luciferase assay demonstrated that the activation domain is essential and the ID enhances activation about 8-fold (FIG. 1B). Nuclear localization did not dramatically enhance activation because the ATF is 23 kDa, small enough to diffuse through the nuclear pore passively (Wang and Brattain, 2007); however, we retained the NLS in the architecture to maximize the ATF's level of induction. When the ATF is too large to passively diffuse into the nucleus, an NLS is advantageously provided in the ATF. Because we wanted to create an ATF library of high complexity, which was as comprehensive as practically possible, it was necessary to determine the minimum number of zinc fingers required to have a transcriptional effect. Toward this end, we compared a two-zinc finger ATF with a three-zinc finger ATF in a luciferase assay (FIG. 8A). The two-zinc finger ATF could only activate the luciferase reporter two-fold over background, while the three-zinc finger ATF was capable of activating 329-fold over background, so we used the three-zinc finger scaffold in the final design.

Next, we identified the in vitro binding preferences for two- and three-zinc finger ATFs with and without the ID. Cognate site identification (CSI) enables the discovery of sequence specificity out of a library of 25-bp sequences (FIG. 8B) (Rodriguez-Martinez et al., 2016; Tietjen et al., 2011). This method involves multiple rounds of enrichment for sequences bound by the DNA-binding factor of interest. The enriched sequences of each round can be multiplexed and sequenced in a high-throughput manner. Rather than focusing on single consensus motifs, the comprehensive binding site specificity of the factor can be displayed in a Specificity and Energy Landscape (SEL) (FIG. 1C). Although the SELs for the ATFs with and without an ID are nearly identical for the EGR1 cognate site (FIG. 8D), there is a set of sequences only targeted by the ATF with the ID (FIG. 1D). Interestingly, the two-zinc finger ATFs target a distinct motif, rather than the expected 5′-(T/G)GG-GCG-3′ site that is bound by the first two fingers of EGR1, emphasizing the dependency of neighboring zinc fingers on binding preferences (Isalan et al., 1997).

To determine how the ATFs impact global transcription, we performed RNA-seq on four ATFs with different architectures. The ATFs either had the first two or all three zinc fingers of EGR1 as the DBD, with and without the ID. Compared to the mock control, the two-zinc finger ATFs had little impact on altering the transcriptional profile (FIGS. 1E, 8E, and 8F). On the other hand, the three-zinc finger ATF with the ID altered the expression of 104 genes (100 upregulated, 4 downregulated) and the three-zinc finger ATF without the ID altered the expression of 57 genes (55 upregulated, 2 downregulated) (FIG. 1E). Most of the genes were upregulated compared to the mock control, and the repressed genes could be attributed to indirect effects of the ATFs. As the three-zinc finger ATF with the ID was capable of binding as a monomer or dimer, most of the subset of genes upregulated by the three-zinc finger ATF without the ID could also be activated by the three-zinc finger ATF with the ID (FIG. 8E). For 27 genes, the ID increased the level of induction triggered by the three-zinc finger ATF. Taking all these results into consideration, the ATF library was created on a scaffold which includes, from N to C terminus: an interaction domain, three zinc fingers with the backbone of EGR1, NLS from EGR1, VP64 activation domain, and a 3× hemagglutinin (HA) tag.

The library was created by incorporating VNN codons, where V is A, C, or G, at the recognition residues (−1, 2, 3, and 6). Use of VNN codons prevents incorporation of premature stop codons within the ORF, and permits the incorporation of 16 different amino acids (FIG. 1F). The library was cloned into a second-generation lentiviral system to ensure efficient delivery to mammalian cells. The ATF is driven by the constitutively active EF-1α promoter, which resists silencing in mammalian cells compared to other constitutive promoters (Teschendorf et al., 2001). The sequence space for all 9-bp sequences is 2.62×10⁵ different sequence permutations. We created an ATF library with a complexity of 2.62×10⁶, ten times the targeted sequence space. Sanger sequencing of 100 clones confirmed success of our design with incorporation of all 16 amino acids at each recognition residue, suggesting diverse representation in the library (FIG. 1F and Table 4).

TABLE 4 Diversity of the ATF library Finger 1 Finger 2 Finger 3 −1 2 3 6 −1 2 3 6 −1 2 3 6 1 A P G S Q L K V V H T R 2 R T R G L Q A Q N D P L 3 K R G T T L P T E M H H 4 G G L R D M A P N H L L 5 L R L Q L K I L I T L T 6 V V R R T P I Q T P V I 7 G T V V E E Q K S L P T 8 I T M G G P T P G E T G 9 M M T G D R P S L D M R 10 G A V A R P I D R I D N 11 T K V E T R V V L T L L 12 M V R T D N N A K I I Q 13 N H R A G A G D L R Q P 14 V G E P L P N I Q E G K 15 V E K K R H R R T H T K 16 G G R P T V Q I R E V H 17 G L E A P S A S Q L I Q 18 D I T I I N D G L V K T 19 R V D T G A L K S T P P 20 P K G R P K N L R N T R 21 R A G H T P I I K E H D 22 K G H V R H P H S T I R 23 E V N Q T N T L A Q P T 24 E P R T L V P T V N T T 25 V A G A A T I Q A A R K 26 A S V I T P G P V R G E 27 T A V E S S M G V P V T 28 V D T V D D E D E R K E 29 T S V G Q A T R P T L D 30 H I V V T T H R T V R Q 31 K R P V T E E P D V H K 32 G N P I P T G E Q P G G 33 R G G G A H L T S A P H 34 R V L T R R S G R I L S 35 R I R I P P P P P L L P 36 A L I T L I L P V N R H 37 K L R V I G S H H H P T 38 I A D A T K A Q N K K T 39 K E I A P N S R H T R H 40 G G P N H P M A P T N L 41 S R G V A R T K L P P A 42 L R P R K Q N N S R P H 43 N G G P H P P P L R E Q 44 P R R K D H G R P R T M 45 K E H G R D A L D T S S 46 L G T L L P T Q I T I N 47 P K T G A G H E Q S T G 48 V D E G T R E R H T L N 49 L D R L D P P A R T D P 50 G R D I N A P P Q I I V 51 V G V P L T Q I K T I T 52 L S L G N T Q S S T I A 53 I L A G M K P T D R R G 54 R R A G D L K R E K E P 55 R R L R T T L V H H K P 56 A L P G H G P L S T A I 57 P R E I I K K P D N I M 58 G M S Q Q P D L K Q K N 59 I R G V R L L R Q T P I 60 R R G G I T P E P L R K 61 I G A G S N Q A R I H R 62 Q R E V N K T T D T Q H 63 K I T A H R I N A P G E 64 R G G E L P L I N R D N 65 G G Q R L H P M K R N T 66 R I Q R P P I T L L T I 67 I T P L T P P L T T M D 68 V S T V P A T N R I T I 69 K L L R V T M L K R V G 70 A L V L H S H H A A G R 71 S Q E A D R I S L P A H 72 K D G G T I A G V G L V 73 R R K P P T K R E P T Q 74 T D R Q P V A K K T K T 75 D L V E T P T Q M L D I 76 I G R R T L P A D Q K I 77 K M T L A N L S K R P S 78 V E P P I V G Q E T I E 79 G L T E Q V A E K R L P 80 V P E G A K N Q D L G I 81 R I P K R P L L T E G R 82 G G R V I P R N T P G T 83 G A I G S P I D A R P G 84 L L A N P R R L T I H S 85 R S M S I I P D H T P T 86 D V R R E A P G P I L V 87 E G G N L R K N T L A S 88 V G G D P R L V S E K A 89 V R V R Q P R G A R S G 90 P K R R K G G V K D L A 91 V M M G I L H Q P P T T 92 L T P V H P A S L A D A 93 G M R I I V A V T I E L 94 P G L K D S L T P E A H 95 N V R V R R P K R G I R 96 I R L M P A P A D E D K 97 R G A A P A K P T P K Q 98 V E I G Q I Q P P I L T 99 E R A P N P A P L R L D 100 P D A R T G T N T I K P

ATF Library can Activate the Pluripotency Network:

We asked whether ATFs in the library could replace the key regulator of pluripotency, Oct4, in the cocktail of transcription factors that triggers the pluripotency network, Oct4, Sox2, Klf4, and c-Myc (OSKM). In order to test a library, capable of sampling thousands of sites in the genome, it was necessary to have a robust read-out of positive phenotypes (FIG. 2A). Toward this end, we used mouse embryonic fibroblasts (MEFs) isolated from a transgenic mouse line that allows lineage tracing of endogenous Oct4 transcription (FIG. 2B) (Greder et al., 2012). In these cells, tamoxifen-inducible Cre recombinase (MerCreMer) is expressed when the endogenous pluripotency associated gene, Oct4, is transcribed. In the presence of 4-hydroxytamoxifen, the recombinase removes Tomato from the ROSA locus, and transmembrane-bound GFP is expressed. Consequently, Tomato⁺GFP⁻ MEFs become Tomato⁻GFP⁺ cells after endogenous Oct4 is activated, and GFP expression is maintained in all their cell progeny.

The ATF library was transduced in MEFs (MOI=3) with Sox2, Klf4, and c-Myc (SKM). As a positive control, we delivered OSKM to MEFs (FIG. 9A). To account for reprogramming events induced by SKM, alone, we delivered lentivirus with an empty ORF in place of the ATF. The lentivirus with the empty ORF accounted for false positive events that could arise from lentiviral delivery or integration of a strong constitutive promoter at relevant genomic sites. We also included a condition with the ATF library alone, as well as untreated MEFs. The lentiviruses encode a puromycin resistance gene, used for selection of cells with integration events. After 15 days in culture with ES cell media, Tomato⁻GFP⁺ cells were sorted by flow cytometry. Compared to OSKM, MEFs treated with the ATF library underwent significant cell death. Among those that survived, 0.229% of the ATF library+SKM cells were Tomato⁻GFP⁺, compared to 0.033% for OSKM, and 0% for untreated MEFs (FIG. 2C). No Tomato⁻GFP⁺ cells were observed in the cells treated with ATF library alone, or Empty+SKM control. Tomato⁻GFP⁺ cells from the condition of ATF library+SKM were isolated as single cells for further analysis. Of the ATF library+SKM cells, a small fraction (0.8%) of cells were Tomato⁺GFP⁺. Because the half-life of Tomato fluorescent protein is ˜24 hours, there is a period of time after Oct4 is activated when the cells are double positive. We sorted these cells separately to determine whether the ATFs expressed in the double positive cells were different from those expressed in the Tomato⁻GFP⁺ cells.

Since multiple ATFs can potentially act in concert to activate the pluripotency network, we identified the ATFs from single cells to capture ATF combinations that had been sufficient, in concert with exogenous SKM, to activate endogenous Oct4 transcription and induce GFP expression. Preliminary evaluation of iPS colonies derived from the screen with mixed combinations of ATFs showed high levels of pluripotency markers (FIG. 9C). Eleven isolated GFP⁺ cells were subjected to two-step nested PCR of genomic DNA, and sequencing results revealed 11 unique combinations of ATFs (FIG. 2D and Table 5). The range of ATFs varied between two and ten ATFs within a single cell. ZFATF1 (blue) and ZFATF2 (orange) appeared in most of the combinations; however, an additional ATF was necessary for conversion to a bona fide iPS cell state. One ATF from cells #4-11 obtained a frameshift mutation near the N-terminus, resulting in a 163-aa protein product that does not code for a zinc finger protein (ZFATF5). Only the interaction domain, as well as the first 19 amino acids of the first zinc finger, remain intact. Protein homology at the primary structure revealed that the ATF with the frameshift mutation has low homology to zinc finger protein 3-like from Gorilla gorilla (G. gorilla); however, the homology occurs not in the DBD of the G. gorilla protein, but in the region C-terminal to the DBD (FIG. 9B). Homology to this uncharacterized transcription factor suggests that the ATF with the frameshift mutation retains a functional role, although the mechanism of regulating gene expression is not obvious from the primary structure.

TABLE 5 Experimental conditions with trial numbers. iPS cells Condition Alias Trials generated Lentivirus Retrovirus 1 OSKM 5 yes N/A Oct4, Sox2, Klf4, (n = 5) c-Myc 2 Empty + SKM 5 no Empty Sox2, Klf4, c-Myc 3 C2 + SKM 5 yes ZFATF1, ZFATF2, ZFATF3 Sox2, Klf4, c-Myc (n = 5) 4 C3 + SKM 5 yes ZFATF1, ZFATF2, ZFATF4 Sox2, Klf4, c-Myc (n = 5) 5 C4 + SKM 5 yes ZFATF1, ZFATF2, ZFATF5 Sox2, Klf4, c-Myc (n = 5) 6 ZFATF1 + 3 no ZFATF1, ZFATF2 Sox2, Klf4, c-Myc ZFATF2 + SKM 7 ZFATF1 + 3 no ZFATF1, ZFATF3 Sox2, Klf4, c-Myc ZFATF3 + SKM 8 ZFATF1 + 3 yes ZFATF1, ZFATF4 Sox2, Klf4, c-Myc ZFATF4 + SKM (n = 1) 9 ZFATF1 + 1 no ZFATF1, ZFATF5 Sox2, Klf4, c-Myc ZFATF5 + SKM 10 ZFATF2 + 3 no ZFATF2, ZFATF3 Sox2, Klf4, c-Myc ZFATF3 + SKM 11 ZFATF2 + 3 no ZFATF2, ZFATF4 Sox2, Klf4, c-Myc ZFATF4 + SKM 12 ZFATF2 + 2 no ZFATF2, ZFATF5 Sox2, Klf4, c-Myc ZFATF5 + SKM 13 ZFATF1 + 3 no ZFATF1 Sox2, Klf4, c-Myc SKM 14 ZFATF2 + 3 no ZFATF2 Sox2, Klf4, c-Myc SKM 15 ZFATF3 + 3 no ZFATF3 Sox2, Klf4, c-Myc SKM 16 ZFATF4 + 3 no ZFATF4 Sox2, Klf4, c-Myc SKM 17 ZFATF5 + 1 no ZFATF5 Sox2, Klf4, c-Myc SKM 18 C2 + SK 1 no ZFATF1, ZFATF2, ZFATF3 Sox2, Klf4 19 C2 1 no ZFATF1, ZFATF2, ZFATF3 N/A 20 C3 + SK 3 no ZFATF1, ZFATF2, ZFATF4 Sox2, Klf4 21 C3 1 no ZFATF1, ZFATF2, ZFATF4 N/A 22 C4 + SK 3 no ZFATF1, ZFATF2, ZFATF5 Sox2, Klf4 23 C4 3 no ZFATF1, ZFATF2, ZFATF5 N/A 24 C1 + SKM 5 no ZFATF6, ZFATF7 Sox2, Klf4, c-Myc 25 C5 + SKM 5 no ZFATF4, ZFATF5, ZFATF8 Sox2, Klf4, c-Myc 26 C6 + SKM 3 no ZFATF1, ZFATF2, ZFATF5, Sox2, Klf4, c-Myc ZFATF6 27 C7 + SKM 3 no ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, c-Myc ZFATF5, ZFATF6, ZFATF7 28 C8 + SKM 2 no ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, c-Myc ZFATF5, ZFATF6, ZFATF7, ZFATF9 29 C9 + SKM 2 no ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, c-Myc ZFATF5, ZFATF6, ZFATF8, ZFATF9, ZFATF11, ZFATF12 30 C10 + SKM 2 no ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, c-Myc ZFATF4, ZFATF5, ZFATF6, ZFATF7, ZFATF9, ZFATF10 31 C11 + SKM 2 no ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, c-Myc ZFATF5, ZFATF6, ZFATF7, ZFATF8, ZFATF9, ZFATF10, ZFATF11 32 C1 + SK 3 no ZFATF6, ZFATF7 Sox2, Klf4 33 C1 1 no ZFATF6, ZFATF7 N/A 34 C5 + SKM 1 no ZFATF4, ZFATF5, ZFATF8 Sox2, Klf4 35 C5 1 no ZFATF4, ZFATF5, ZFATF8 N/A 36 ZFATF1 1 no ZFATF1 N/A 37 ZFATF2 1 no ZFATF2 N/A 38 ZFATF5 1 no ZFATF5 N/A 39 ZFATF1-5 + 2 no ZFATF1, ZFATF2, ZFATF3, Oct4, Sox2, c-Myc SKM ZFATF4, ZFATF5 40 C2 + OSKM 1 yes ZFATF1, ZFATF2, ZFATF3 Oct4, Sox2, Klf4, (n = 1) c-Myc 41 C3 + OSKM 1 no ZFATF1, ZFATF2, ZFATF4 Oct4, Sox2, Klf4, c-Myc 42 GFP 5 no N/A GFP

All ATF combinations identified in the screen for endogenous Oct4 expression were re-validated to determine whether they were true positives. Among the eleven ATF combinations, #2, #3, and #4 could generate colonies of iPS cells when expressed with Sox2, Klf4, and c-Myc (FIG. 2E and Exhibit A, Dataset 1). Interestingly, Combination #4 came from the Tomato⁺GFP⁺ cells, in which Oct4 was activated before the Tomato signal dissipated. During the validation step, MEFs expressing ATF Combination #4+SKM became iPS cells ˜28 days later than the iPS cells generated by the other ATF combinations or OSKM. The doubling times for the iPS cells generated with Combinations #2 and #3 were comparable to that of iPS cells generated with OSKM; however, the doubling time for iPS cells expressing Combination #4 was slightly longer (FIG. 9D). iPS cells generated with ATFs demonstrated capacity for self-renewal and have been cultured beyond 65 passages.

iPS Cells Generated with ATFs are Pluripotent:

iPS cells generated with ATFs were further characterized for markers of pluripotency. Immunofluorescence was performed to confirm expression of pluripotency markers, OCT4, SOX2, and NANOG (FIGS. 3A and 10A). To determine whether the iPS cells generated with ATFs were functionally pluripotent, we generated embryoid bodies and measured markers of the germ layers by immunofluorescence and RT-qPCR. Immunocytochemistry was used to detect myosin light polypeptide 2 (mesoderm), forkhead box A2 (endoderm), and beta III tubulin (ectoderm) in outgrowths from the embryoid bodies (FIGS. 3B and 10B). Furthermore, RT-qPCR results for brachyury, NK2 homeobox 5, and kinase insert domain receptor (mesoderm); alpha fetoprotein, transthyretin, and forkhead box A2 (endoderm); nestin, neurofilament light polypeptide, and SRY-box 17 (ectoderm) confirmed differentiation into all three germ layers (FIGS. 3C, 3D, 10C, and 10D). Compellingly, beating cardiomyocytes were observed in embryoid outgrowths from EBs derived from iPS cells generated by ATFs. Taken together, the embryoid body data suggests that the iPS cells generated with ATFs are functionally pluripotent.

From morphological and select gene marker analysis, we focused on global transcriptome analysis of the ATF-treated cells. Comparison of genome-wide transcriptional profiles showed that the iPS cells generated with ATFs cluster with mouse ES cells and iPS cells generated with OSKM (FIGS. 4A and 11A). Cells at early stages of reprogramming clustered with MEFs and Empty+SKM. iPS cells generated with ATFs show an upregulation of pluripotency markers and a downregulation of fibroblast markers (FIG. 4B). Using the 853 genes that make up the fibroblast gene regulatory network (GRN) and the 705 genes that make up the pluripotency GRN from CellNet (Cahan et al., 2014), we compared the expression profiles of iPS cells generated with ATFs to those of other pluripotent cells and MEFs. Our genome-wide analysis indicates our ATF-induced iPS cells display a high degree of similarity with pluripotent cells generated using exogenous retrovirally-delivered Oct4. It is important to note that, at early stages of reprogramming, ATF-treated cells have a different profile compared to OSKM-treated cells (FIGS. 4C and 4D). These differences suggest other underlying regulators beyond what is characterized in the GRNs of CellNet guide cells to pluripotency (FIG. 4D). Once fully reprogrammed to the pluripotent state, global transcriptional profiling shows iPS cells generated with ATFs share more similarity amongst themselves than to OSKM or ES cells (FIGS. 4A and 11C). ATF levels at early stages of reprogramming were detectable; however, once converted to iPS cells, the lentiviral elements controlling the expression of ATFs are silenced, a further confirmation that the cells were fully reprogrammed with the endogenous pluripotency circuitry activated.

Signature Epigenetic Landscapes at ATF-Activated Pluripotency Genes:

The chromatin structure of iPS cells generated with ATFs was compared to those generated with OSKM. Specifically, ChIP-seq was performed on histone 3 lysine 27 acetylation (H3K27ac), a marker for super-enhancers (Hnisz et al., 2013; Whyte et al., 2013), and histone 3 lysine 9 trimethylation (H3K9me3), a marker of heterochromatic repressed regions of chromatin. The pluripotent cells shared similar sets of peaks for H3K27ac regardless of whether they were generated with ATFs or with natural factors (FIGS. 5A and 5B). Likewise, H3K9me3 peaks were similar for ATF-generated iPS cells and OSKM-generated iPS cells, although there was greater overlap for OSKM and Combination #3+SKM (FIGS. 6A and 6B). There was more similarity between OSKM-generated iPS cells and iPS cells generated with Combination #3+SKM. Comparison of the super-enhancer peaks of the iPS cells in this study to those of MEFs in the mouse ENCODE data revealed the greatest number of differences between MEFs to iPS cells and the greatest number of overlaps occurred among the iPS cells generated with ATFs+SKM and OSKM (FIGS. 5A and 5B). Although mouse ENCODE data for H3K9me3 was not available, both repressive and active histone marks show very strong correlations among the iPS cells induced by ATFs+SKM and by OSKM (FIGS. 5C and 6C).

ATF-Triggered Networks:

The ATF targets were examined by integrating Cognate Site Identification (CSI) sequence energy landscapes of ATFs with global transcription obtained by RNA-seq (FIG. 7A). The preferred binding sites of the ATFs were determined by CSI (FIG. 13A). To pinpoint the genes in the pluripotency network activated by the ATFs, the top five binding motifs for each ATF were determined bioinformatically. Binding sites within ±1 kb of the transcriptional start site (TSS) were considered in the analysis. We chose this narrow window based on ATF design principles (Rebar et al., 2002), the tendency of sequence-specific TFs to exhibit a peak −300 bp relative to the TSS (Koudritsky and Domany, 2008), and evidence that the predictive power of TF binding on gene regulation drops significantly when the binding sites examined are beyond 2 kb from the TSS (Cheng and Gerstein, 2012; Whitfield et al., 2012). Genes, expressed >2-fold more in iPS cells generated from ATFs than Empty+SKM cells and were statistically significant, were filtered with the gene list with ATF binding sites within ±1 kb of the TSS. Two other pairwise comparisons (ATF Combination #2/3/4+SKM at the early stage versus iPS stage and ATF Combination #2/3/4+SKM iPS cells versus OSKM iPS cells) were included in the analysis. Finally, those genes, upregulated >2-fold and exhibited ATF binding sites, were further examined for being implicated in inducing pluripotency from previous studies (FIG. 7B and Exhibit A, Dataset 2) (Buganim et al., Cell 150(6): 1209-1222, 2012; Heng et al., Cell Stem Cell 6(2):167-174, 2010; Kim et al., Cell 132(6):1049-1061, 2008; Krentz et al., Dev Biol 377(1):67-78, 2013; Lujan et al., Nature 521(7552):352-356, 2015; Marson et al., Cell 134(3):521-533, 2008; Sharov et al., BMC Genomics 9(1):269, 2008; Shu et al., Cell 153(5):963-975, 2013; Som et al., PLoS ONE 5(12):e15165, 2010). The super-enhancer profiles for the ATF target genes show H3K27ac peaks that confirm that they are actively expressed (FIG. 7C). A gene regulatory network based on the CSI results and differential expression data was built using information from the literature and the STRING database (FIG. 7D).

Next, we expanded our analysis to include high and medium affinity binding sites from CSI data of Combination #2. We used the top 100-scoring 10-bp motifs to identify genes, potentially targeted by the ATFs (FIG. 13B). The target genes were not filtered with expression data in this comprehensive analysis of the CSI data. Gene set enrichment analysis with Enrichr (Chen et al., 2013) for 2897 genes with a sum CSI score of 20 or greater, in other words, six or more ATF target sites within ±1 kb of the TSS showed an overrepresentation of genes found in PluriNetWork for M. musculus (FIG. 7E) (Som et al., PLoS ONE 5(12):e15165, 2010). This analysis was then performed for Combinations #3 and #4, and similar results were obtained, suggesting that the different ATF combinations activate similar nodes of the pluripotency network.

Surprisingly, our data demonstrates that Oct4 does not appear to be the primary target as it ranks 4788 for the sum CSI score for Combination #2, 3929 for Combination #3, and 5225 for Combination #4. Other genes with higher sum CSI scores would be targeted by ATFs directly, and those that are key regulators in the pluripotency circuitry, subsequently, trigger the activation of Oct4. A comparison of Oct4 targets with the ATF targets reveals striking differences (FIGS. 7D and 12). Thus, Oct4 is indirectly activated by the ATFs, and the path the MEFs take to reprogram to the iPS state is different than the path taken when treated with OSKM.

A gene regulatory network based on the CSI results and differential expression data was built using information from the literature and the STRING database (FIG. 15A) (Szklarczyk D, et al. (2015) Nucleic Acids Res 43(Database issue):D447-52). Greater ATF occupancy near the TSS for the 17 predicted targets in the gene regulatory network suggests they are direct targets (FIG. 15B). A comparison of direct OCT4 targets with the ATF target genes reveals striking differences (FIG. 15C) (Som A, et al. (2010) PLoS ONE 5(12):e15165).

These differences suggest that ATFs activate the pluripotency network through different nodes than the exogenously expressed Oct4. While the primary targets may differ at the outset, the eventual iPS cells show remarkable convergence in the transcriptome profiles and epigenetic landscapes.

Discussion

Zinc finger, TAL effector, and CRISPR/Cas9 libraries have been tested for loss-of-function phenotypes, acquisition of resistance to a drug, or upregulation of specific genes (Bae et al., 2003; Blancafort et al., 2003; Kim et al., 2013; Konermann et al., 2015; Li et al., 2014; Tschulena et al., 2009; Wang et al., 2014; Zhou et al., 2014); however, this is the first report of a gain-of-function screen to reprogram fibroblasts to iPS cells, a feat that requires drastic transcriptional and epigenetic changes. We screened a zinc finger library of high complexity, not previously tested in mammalian cells. Furthermore, the capacity for the ATFs to cooperatively bind target genes provides the ATF library with a unique feature to sample a larger set of binding sites and activate to greater extent due to synergy. Previous, conventional zinc finger libraries consisted of ATFs created by shuffling ˜55 zinc finger units, previously characterized to bind specific triplets of nucleotides (Gonzalez et al., 2010). The library used in this study uses a much larger repertoire of residues, incorporating 16 of the 20 possible amino acids in the recognition residues, greatly expanding the target space of the ATFs. A survey of natural zinc fingers found in eukaryotes reports that all amino acids are represented in the recognition residues (Najafabadi et al., 2015).

Because we are using a large library of ATFs, capable of sampling thousands of genes in parallel, there is a potential for our ATFs to activate endogenous Oct4 directly; however, in our analysis, we find that the ATFs seem to activate Oct4 indirectly. ChIP-seq of the ATFs was not possible due to silencing of the lentiviral elements in iPS cells. Although ChIP-seq at an earlier stage before full conversion can provide insight on the genomic targets of the ATFs, the signal for functionally relevant ATF binding sites would be challenging to detect due to low frequency of conversion of parental fibroblasts to iPS cells.

Application of the ATF library in this gain-of-function screen demonstrates that zinc finger ATFs can perturb the transcriptional profile of a cell to levels that are sufficiently robust to induce a dramatic phenotypic change. Like natural TFs, the ATFs in this study target 9-10 bp sites and can bind cooperatively as homodimers and heterodimers to a much larger and perhaps less frequent 18 bp site. As each ATF in the library will have unique sequence preferences and varying degrees of affinity for DNA, a wide range of outcomes can be elicited upon introduction of these ATFs. Unlike natural TFs, ATFs do not necessarily rely on partner proteins, and thus, transcriptional networks can be stimulated from any homeostatic state.

In summary, this study provides a compelling support for our design principles and demonstrates unequivocally that an ATF library can be used in a gain-of-function screen for different cell fate conversions. Interestingly, during the early stages of conversion, cells expressing ATFs+SKM exhibited a different transcriptional profile from those expressing OSKM; however, in the final iPS cell state, the expression profiles of all the pluripotent cell types were similar both at the molecular and functional levels. The differences in molecular signatures at the early stage suggest that the MEFs take different dedifferentiation routes to the same pluripotent state.

In addition to providing a means to perform a forward genetic screen, this Example describes a strategy for identifying cell-fate defining transcriptional networks. By integrating expression data with in vitro binding site data, we were able to identify the nodes of the transcriptional network implicated in the induction of pluripotency. Furthermore, ATF libraries can be used to identify unanticipated regulatory networks in an unbiased manner. This technology enables the pursuit of elusive cell phenotypes or direct conversions, considered challenging to achieve by conventional methods.

Example 2—Cardiomyocyte Differentiation Using ATFs

Differentiation into cardiomyocytes is valuable for disease modeling, drug testing, and heart regeneration. Current methods to differentiate human pluripotent stem cells into cardiomyocytes involve temporal modulation the Wnt pathway with small molecule inhibitors. While current methods are efficient for the derivation of ventricular cardiomyocytes, these cardiomyocytes resemble a more fetal phenotype, and robust differentiation into other subtypes, such as atrial cells and nodal cardiomyocytes, remains difficult.

An unbiased, artificial transcription factor (ATF) library-based approach circumvents this challenge and allows for the selection of a particular cell fate. To demonstrate that an ATF library can be used as an unbiased screen for cardiomyocyte differentiation, we tested our gene-activating zinc finger ATF library in which each ATF targets a 9-bp sequence. This library is composed of 2.6×10⁶ ATFs, a complexity that encompasses 10-times the sequence space of all 9-bp permutations. An important and distinguishing feature of our ATF design is the incorporation of an interaction domain that allows two ATFs to dimerize and activate target genes in a synergistic manner. We tested the ATF library in replacing the function of the Wnt pathway inhibitors used to derive cardiomyocytes. Our results suggest that the ATF library can be used to differentiate cells into more challenging subtypes, such as atrial cardiomyocytes.

ATF Library Screening in Cardiomyocyte Differentiation

Directed differentiation to cardiomyocytes involves modulation of the Wnt pathway to induce a step-wise transition to mesodermal progenitor cells, followed by specification into cardiomyocytes (FIG. 18A). First, cells are treated with CHIR99021, a GSK3 inhibitor, resulting in the activation of the Wnt pathway. GSK3 is a kinase that phosphorylates β-catenin and mediates its ubiquitination and subsequent proteasomal degradation. By inhibiting GSK3, β-catenin target genes are activated, inducing specification into mesoderm. The progenitor cells at this point express T (Brachyury). Three days after treatment with the GSK3 inhibitor, the Wnt pathway is suppressed with IWP2 to promote specification into cardiomyocytes. IWP2 is a molecule that inhibits Porcupine, an enzyme that palmitoylates Wnt proteins for secretion. By Day 10 of differentiation, cells express cardiomyocyte-specific genes, TNNT2 (cardiac troponin T2), GATA4, and NKX2.5 at high levels.

We asked if the temporal modulations of the Wnt pathway could be regulated with ATFs. Toward this end, we tested the ATF library in H9 human embryonic stem (ES) cells bearing a GFP reporter for TNNT2. In these H9-cTnnT-pGZ cells, the promoter of TNNT2 drives the expression of GFP and zeocin, permitting the isolation of cells that express the cardiomyocyte-specific marker (FIG. 19B).

The ATF library was cloned into a second generation lentiviral vector. The screen is performed in cells with a robust change in phenotype or a lineage-specific reporter. H9-cTnnT-pGZ cells were transduced with ATFs or lentiviruses without an ORF (Empty control) (FIG. 18B). After selection of integration events with puromycin (FIG. 19A), cells were treated with the Wnt pathway inhibitors at the appropriate time points (FIG. 18A). Positive outcomes were isolated as single cells, such that combinations of ATFs, if any, can be captured. Integrated ATFs were identified from such single cells and retested for validation. Once validated, downstream experiments were be performed to identify ATF target genes. For example, by omitting the GSK3 inhibitor (CHIR), the Wnt inhibitor (IWP2), or both, we asked whether ATFs could replace the function of these inhibitors. ATFs were able to replace the function of the Wnt inhibitor, IWP2 (FIG. 18C).

After treatment with the GSK3 inhibitor, these cells could differentiate into cardiomyocytes and express the GFP reporter for TNNT2 (FIG. 19C). Flow cytometry of ATF-treated cells resulted in 20.8% of cells expressing GFP compared to 1.1% for the Empty control. Differentiation into cardiomyocytes in the Empty control could be attributed to spontaneous differentiation, introduction of a strong promoter (EF1α) to a locus proximal to a positive regulator of cardiomyocyte differentiation, disruption of a negative regulator of cardiomyocyte differentiation from lentiviral integration, or activation of genes in response to delivery of lentivirus. The Empty control demonstrated the rate of expected false positive events to be 5.3%. By cell sorting, we isolated the GFP⁺ cells as single cells (FIG. 19A). From 16 single cells, we found 10 unique combinations of ATFs capable of replacing the function of the Wnt inhibitor, IWP2 (FIG. 19D and Table 6). One combination occurred in two cells and another combination in six cells. ATF5 and ATF1 appeared in every cell except one. Due to the recurring nature of Combination #3-8 (C3+CHIR), we focused on retesting this combination of ATFs as well as subsets of this combination.

TABLE 6 Sequences of ATF Hits in Cardiomyocyte Differentiation ATF DNA sequence ZFATF1 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGTGATCGCCGCTTCTCCGTTTCC CACCGGCTCACCCATCACATCCGCATCCACACAGGCCAGA AGCCCTTCCAGTGCCGCATCTGCATGCGCAACTTCAGCCCT AGCAGAAGACTCACCCAGCACATCCGCACCCACACAGGCG AAAAGCCCTTCGCCTGCGACATCTGTGGAAGAAAGTTTGC CCAGAGCGAGGGGCGCAAGGTCCATACCAAGATCCACTTG CGGCAGAAGGACAAGAAAGCAGACAAAAGTGTTGTGGGG CGCGCCGACGCGCTGGACGATTTCGATCTCGACATGCTGG GTTCTGATGCCCTCGATGACTTTGACCTGGATATGTTGGGA AGCGACGCATTGGATGACTTTGATCTGGACATGCTCGGCT CCGATGCTCTGGACGATTTCGATCTCGATATGTTAATTAAC TACCCGTACGACGTTCCGGACTACGCTGGTTATCCCTATGA CGTCCCGGATTATGCAGGATCCTATCCATATGACGTTCCAG ATTACGCTTGA (SEQ ID NO: 72) ZFATF2 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCGGGTCCGTAGTCCTCACCAAACACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCATAAGCACTGTTCTCACCGGTCACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCAACAGCGAGCCACGCAAGACCC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 73) ATF5 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGATTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCAGATCCCATCGGCCACCAGCCACATCCGCAT CCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCATG CGCAACTTCAGCGTCAGCCCGACCCTCACCCGACACATCC GCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCTG TGGAAGAAAGTTTGCCAGGAGCGACCAGCGCAAGAGACA TACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAGA CAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTTC GATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTGA CCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGAT CTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATCT CGATATGTTACCCGTACGACGTTCCGGACTACGCTGGTTAT CCCTATGACGTCCCGGATTATGCAGGATCCTATCCATATGA CGTTCCAGATTACGCTTGA (SEQ ID NO: 76) ZFATF6 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGTGATCGCCGCTTCTCCGTTTCC CACCGGCTCACCCATCACATCCGCATCCACACAGGCCAGA AGCCCTTCCAGTGCCGCATCTGCATGCGCAACTTCAGCCTC AGCCTAACCCTCACCAAACACATCCGCACCCACACAGGCG AAAAGCCCTTCGCCTGCGACATCTGTGGAAGAAAGTTTGC CAAAAGCAAACCACGCAAGACACATACCAAGATCCACTTG CGGCAGAAGGACAAGAAAGCAGACAAAAGTGTTGTGGGG CGCGCCGACGCGCTGGACGATTTCGATCTCGACATGCTGG GTTCTGATGCCCTCGATGACTTTGACCTGGATATGTTGGGA AGCGACGCATTGGATGACTTTGATCTGGACATGCTCGGCT CCGATGCTCTGGACGATTTCGATCTCGATATGTTAATTAAC TACCCGTACGACGTTCCGGACTACGCTGGTTATCCCTATGA CGTCCCGGATTATGCAGGATCCTATCCATATGACGTTCCAG ATTACGCTTGA (SEQ ID NO: 77) ZFATF11 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCATATCCGGTGCGCTCACCGGGCACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCAGTAGCAATCAACTCACCGCGCACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCAGAAGCATACACCGCAAGCGTCA TACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAGA CAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTTC GATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTGA CCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGAT CTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATCT CGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 82) ZFATF13 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGTGATCGCCGCTTCTCCGTTTCC CACCGGCTCACCCATCACATCCGCATCCACACAGGCCAGA AGCCCTTCCAGTGCCGCATCTGCATGCGCAACTTCAGCCCT AGCAGAAGACTCACCCAGCACATCCGCACCCACACAGGCG AAAAGCCCTTCGCCTGCGACATCTGTGGAAGAAAGTTTGC CAGGAGCGACCAGCGCAAGAGACATACCAAGATCCACTT GCGGCAGAAGGACAAGAAAGCAGACAAAAGTGTTGTGGG GCGCGCCGACGCGCTGGACGATTTCGATCTCGACATGCTG GGTTCTGATGCCCTCGATGACTTTGACCTGGATATGTTGGG AAGCGACGCATTGGATGACTTTGATCTGGACATGCTCGGC TCCGATGCTCTGGACGATTTCGATCTCGATATGTTAATTAA CTACCCGTACGACGTTCCGGACTACGCTGGTTATCCCTATG ACGTCCCGGATTATGCAGGATCCTATCCATATGACGTTCCA GATTACGCTTGA (SEQ ID NO: 92) ZFATF14 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCCGTTCCATCCGTCTCACCATACACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCGGTAGCGTTCAACTCACCATACACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCCACAGCGAACCTCGCAAGACACA TACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAGA CAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTTC GATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTGA CCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGAT CTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATCT CGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 93) ZFATF15 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCGGGTCCAGCGGGCTCACCATGCACATCCGC ATCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCA TGCGCAACTTCAGCCTAAGCACAACACTCACCAACCACAT CCGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATC TGTGGAAGAAAGTTTGCCACAAGCCGTACACGCAAGCGGC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 94) ZFATF16 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCCGTTCCATCCGTCTCACCATACACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCCACAGCCCGCAACTCACCCCTCACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCGAAAGCCGTAAACGCAAGGAAC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 95) ZFATF17 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCCGTTCCATCCGTCTCACCATACACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCGGTAGCGTTCCACTCACCATACACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCAGGAGCGACCAGCGCAAGAGAC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 96) ATF18 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGCCCTGTGATCG CCGCTTCTCCCGTTCCATCCGTCTCACCATACACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCAGCATCTGCAT GCGCAACTTCAGCGGTAGCGATCAACTCACCATAAACAAC CGCACCAACACAGGCGAAAAGCCATACGCATGA (SEQ ID NO: 97) ZFATF19 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCATATCCGGTGCGCTCACCGGGCACATCCGCA TCCACACAGGCCAGAAGCCCTTCGCCTGCGACATCTGTGG AAGAAAGTTTGCCAGGAGCGACCAGCGCAAGAGACATAC CAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAGACAA AAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTTCGAT CTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTGACCT GGATATGTTGGGAAGCGACGCATTGGATGACTTTGATCTG GACATGCTCGGCTCCGATGCTCTGGACGATTTCGATCTCGA TATGTTAATTAACTACCCGTACGACGTTCCGGACTACGCTG GTTATCCCTATGACGTCCCGGATTATGCAGGATCCTATCCA TATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 98)

Compared to the positive control in which H9-cTnnT-pGZ cells are treated with both inhibitors (CHIR and IWP2), C3+CHIR could generate cardiomyocytes just as efficiently. We also qualitatively assessed the quality of the cardiomyocytes by evaluating their capacity to beat and form large cardiomyocyte sheaths. The cardiomyocytes derived from C3+CHIR formed large beating sheaths with a strong, regular beat, demonstrating functional changes in phenotype. Subsets of C3+CHIR that also generated healthy cardiomyocytes, include Treatment 7, Treatment 8, and Treatment 9 (FIG. 20). Compared to the positive control, cells expressing two or more ATFs generated cardiomyocytes of equal quality. Treatment 9 produced cardiomyocytes at a high efficiency, equal to or greater than the positive control. Treatment 6 demonstrated that one ATF was sufficient to replace the function of IWP2; however, the cardiomyocyte sheets were narrower for this treatment. The Empty control accounts for spontaneous differentiation due to lentiviral delivery or random integration of a strong promoter at a relevant site in the genome. The differentiated cells in Treatments 3, 4, and 5 were morphologically different from the cardiomyocytes in the positive control despite being GFP and exhibited irregular beating. Of the subsets, Treatment 9 (ATF5+ZFATF2+CHIR) most efficiently generated cardiomyocytes. When ATF5 was expressed alone with CHIR treatment (Treatment 9), we were able to generate cardiomyocytes; however, narrower sheaths formed, suggesting lower efficiencies in conversion. Other subsets, including ATFs expressed one at a time, did not generate healthy cardiomyocytes with a regular beat.

ATF Targets and Mechanism of Action

In order to identify the ATF target genes, cognate site identification (CSI) described in Chapter 3, was used to find the top 100 scoring motifs for each ATF. The locations of these top-scoring motifs were identified in the human genome, then annotated to the nearest genes. The frequency of occurrence in the genome was plotted as well as the frequency within ±1 kb of a transcription start site (TSS) (FIGS. 21A-21B). Table 6 presents the frequency of occurrence of ATF binding sites for the top 100 scoring CSI motifs. ATFs: The ATFs of C3+CHIR. Total: The number of ATF binding sites in the human genome for the top 100 scoring CSI motifs. Near TSS: The number of ATF binding sites within ±1 kb of a TSS. Genes: The number of genes with ATF binding sites within ±1 kb of a TSS. Cardiomyocyte: The number of cardiomyocyte genes with ATF binding sites within ±1 kb of a TSS. Early: The number of cardiomyocyte genes with ATF binding sites within ±1 kb of a TSS expressed at an early stage. Middle: The number of cardiomyocyte genes with ATF binding sites within ±1 kb of a TSS expressed at a middle stage. Late: The number of cardiomyocyte genes with ATF binding sites within ±1 kb of a TSS expressed at a late stage of differentiation. Cardiomyocyte markers: see Burridge et al., 2012 Cell Stem Cell 10(1): 16-28; Stillitano et al., 2012 Drug Discov Today 9(4):e229-e236.

Although there are more than 370,000 medium to high affinity binding sites in the genome, fewer than 10,000 are near a TSS (Table 7). Among those that are near a TSS, 30 genes are markers of cardiomyocytes for C3+CHIR. ATF5 targets the greatest number of cardiomyocyte genes, consistent with the result that ATF5+CHIR can generate cardiomyocytes, albeit at a lower efficiency than C3+CHIR. Also consistent with the bioinformatic analysis is that a larger fraction of ATF binding sites for Treatment 9 (ATF5+ZFATF2+CHIR) occur for late genes, rather than early ones, compared to C3+CHIR, increasing the efficiency of conversion to cardiomyocytes from mesodermal progenitor cells (FIGS. 22A-22C).

TABLE 7 ATF binding site summary Near ATFs Total TSS Genes Cardiomyocyte Early Middle Late ATF5 374,143 9,197 7,514 16 5 4 7 ZFATF1 468,693 9,889 7,925 15 5 6 4 ZFATF2 477,041 9,872 7,811 12 1 2 9 ATF5 + ZFATF1 + 1,319,877 28,958 16,654 30 8 8 14 ZFATF2 ATF5 + ZFATF1 842,836 19,086 12,949 25 8 7 10 ATF5 + ZFATF2 851,184 19,069 12,861 24 6 5 13

Discussion

This study demonstrates that an ATF library can be used as an effective forward genetic screen to identify transcriptional regulators of cardiomyocyte differentiation. By testing the ATF library in the context of replacing the function of the Wnt pathway inhibitors, we were able to show that ATFs can promote differentiation into cardiomyocytes after conversion to mesodermal progenitor cells. By developing a high throughput strategy of sequencing ATFs, the ability to identify the ATFs responsible for cardiomyocyte differentiation from single cells is much more straightforward. Bioinformatic analysis of CSI motifs suggests that the ATFs target cardiomyocyte-specific genes. Additional analysis on upstream targets by chromatin immunoprecipitation as well as further characterization of the ATF-generated cardiomyocytes would aid in the understanding of how the ATFs induce differentiation. Comparisons of cardiomyocyte-specific transcripts would allow us to determine the contribution of the ATFs in inducing differences in quality of cardiomyocytes and efficiency of conversion.

Materials and Methods

Cell Culture:

H9 cTnnT-pGZ cells were maintained in E8 (DMEM/F12 with HEPES, 64 mg/L L-ascorbic acid, 14 μg/L sodium selenium, 100 μg/L FGF2, 19.4 mg/L insulin, 10.7 mg/L transferrin, and 2 μg/L TGFβ1) on Matrigel® substrate (BD Biosciences, NJ). Cells were passaged with 0.5 mM EDTA. For cardiomyocyte differentiation, cells were maintained in RPMI 1640+B27 without insulin for the first 9 days and in RPMI 1640+B27 with insulin after Day 9. Differentiation was performed by addition of CHIR99021 (6 μM) on Day 0 and IWP2 (5 μM) on Day 3. Cells were maintained in a humidified 37° C. incubator with 5% CO₂.

Lentivirus Production:

ATFs and the empty control were packaged into lentivirus using a second-generation lentiviral system. Lentivirus was produced in HEK293T cells by calcium phosphate transfection of pSIN expression, psPAX2 packaging, and pMD2.G envelope plasmids. Media containing virus was harvested 48-60 hours post-transfection. Lentivirus was centrifuged with a sucrose cushion at 25,000 rpm for 2 hours. Viral particles were suspended in PBS and concentrated virus was stored at −80° C. Viral titers were measured by counting HEK293T cells that survived selection after 2 days in media containing puromycin (3 μg/mL).

Lentivirus Transduction:

Lentiviruses were delivered to H9 cTnnT-pGZ cells with 8 μg/mL polybrene. Selection was performed in media containing puromycin (1 μg/mL first day, 1.5 μg/mL second day). The ATF was driven by a constitutive promoter, EF1α.

Flow Cytometry:

Flow Cytometry was conducted for GFP using a FACS Calibur flow cytometer (BD). Cells were sorted into a 96-well plate for single-cell isolation.

Identification of ATFs from Single Cells:

ATF sequences were identified by 3-step PCR followed by high-throughput sequencing. In the first round of PCR, ATFs were amplified with primer set 1 (Table 8). During the third PCR step, amplicons were barcoded with Illumina indexes (Epicentre RSBC10948, SSIP1202, SSIP1203, and SSIP1204). 48 cells were multiplexed on a MiSeq chip with paired-end 250 bp reads.

TABLE 8 Primer Sequences for ATF Identification Primer 5′-3′ Forward 1 TCAAGCCTCAGACAGTGGTTC (SEQ ID NO: 111) Reverse 1 TCACTAATACCTTACTTAACTCTAATCTTAGTCAAGC GTAATCTGGAACGTCATATGGATAGGATCCTGC (SEQ ID NO: 112) Forward 2 TACTATCACTAACTTAGCCGCCACCATG (SEQ ID NO: 113) Reverse 2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTC CTTCTGCCGCAAGTGGAT (SEQ ID NO: 114) Forward 3 AATGATACGGCGACCACCGAGATCTACACTCTTTCCC TACACGACGCTCTTCCGATCTGCTGAACTACGTGGTG CCGAAG (SEQ ID NO: 115) Reverse 3 CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTG GAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 116) X indicates nucleotides from barcodes.

Sequencing data was analyzed by searching for sequences identifying fingers 1, 2, and 3 (Table 9).

TABLE 9 Identifying Sequences Region 5′-3′ 5′ zinc finger 1 TGTGATCGCCGCTTCTCC (SEQ ID NO: 117) 3′ zinc finger 1 CTGTGTGGATGCGGATGTG (SEQ ID NO: 118) 5′ zinc finger 2 TGCATGCGCAACTTCAGC (SEQ ID NO: 119) 3′ zinc finger 2 TTCGCCTGTGTGGGTGCGGATGTG (SEQ ID NO: 120) 5′ zinc finger 3 TGTGGAAGAAAGTTTGCC (SEQ ID NO: 121) 3′ zinc finger 3 CGCAAGTGGATCTTGGTATG (SEQ ID NO: 122)

Microscopy:

Micrographs were taken with a 10× objective on a Zeiss Observer A1 with an AxioCam ICm1.

Cognate Site Identification:

HEK293 cells were transiently transfected with ATFs using Lipofectamine 2000 (Thermo Fisher #11668019). Cells that underwent a mock transfection (without an expression plasmid for ATFs) served as a negative control. Cells were harvested 48 hours post-transfection. Lysates were prepared by lysing 10⁷ cells in 300 μL of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, and 0.1% Na deoxycholate). A 21.5 G syringe needle was used for mechanical lysis. Lysates were centrifuged at 10 k×g for 10 min to separate precipitated genomic DNA from protein constituents. The supernatant, containing ATF protein, was used for CSI. HA magnetic beads (MBL #M132-9) were added to the cell lysates for immunoprecipitation. The binding reaction between the ATFs and 100 nM synthetic DNA was performed in binding buffer (25 mM HEPES, 80 mM KCl, 0.2 mM EDTA, 1 mM MgCl₂, 0.1 mM ZnSO₄) at 25° C. After pull-down, three quick washes with 100 μL ice-cold binding buffer were performed to remove unbound DNA. Magnetic beads were resuspended in a PCR master mix (Lucigen #30035-1) and the DNA was amplified for 15 or 18 cycles. Amplified DNA was column purified (Qiagen #28106), and this enriched DNA pool was used for the subsequent round of enrichment, for a total of three rounds. After three rounds of selection, Illumina sequencing adapters and a unique 6 bp barcode for multiplexing were added by PCR amplification. The starting library (Round 0) was also barcoded. Up to 180 samples were combined and sequenced in a single HiSeq2000 lane.

Illumina sequencing yielded ˜180 million reads per lane. Reads were de-multiplexed by requiring an exact match to the 6 bp barcode and truncated to include only the 25 bp derived from the random portion of the library. On average, we obtained 709,300 reads per barcode. The occurrence of every k-mer (lengths 8 through 16 bp) was counted using a sliding window of size k. To correct for biases in our starting DNA library, we took the ratio of the counts of every k-mer to the expected number of counts in the mock-transfected control. The mock control was modeled using a 5th-order Markov Model derived from the sequencing reads corresponding to the starting library (Round 0). We then calculated a Z-score=(x−μ)/σ or (CSI score minus mean)/standard deviation for each k-mer, using the distribution of k-mer enrichment values (CSI score) for the ATF.

Bioinformatic Analysis of ATF Binding Sites:

The genomic locations of the top five or 100 high-scoring 10-bp motifs from CSI were identified in the mm10 genome by the findMotif utility from the UCSC Genome Browser. These genomic sites were annotated using Homer. Annotated sites were filtered to those within +1 kb of the TSS.

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All publications, including but not limited to patents and patent applications, cited below are herein incorporated by reference as though set forth in their entirety in the present application.

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Example 3—Differentiation of Pluripotent Stem Cells into Blood Lineages Using ATFs

Hematopoietic stem and progenitor cells (HSPCs) continually replenish cells of the blood lineage throughout the lifetime of an individual (1). Although fundamental regulators that guide cells toward hematopoietic fates are known, the precise mechanisms by which the largely quiescent pool of HSPCs proliferate then differentiate are not known (2). HSPCs can give rise to all mature blood cells, and the first step of specification occurs as they differentiate to common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs). Hematopoietic stem and progenitor cell (HSPC) give rise to common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs). CMPs differentiate into megakaryocyte/erythroid progenitors and granulocyte/macrophage progenitors, which can differentiate further into specialized cell types of the blood (1).

To overcome these challenges, we created an artificial transcription factor (ATF) library composed of 2.6×10⁶ ATFs, a complexity that encompasses 10-times the sequence space of all 9-bp permutations. An important and distinguishing feature of our ATF design is the incorporation of an interaction domain that allows two ATFs to dimerize and activate target genes in a synergistic manner. We tested the ATF library in inducing differentiation into blood lineages. As demonstrated in this Example, the ATF library can serve as a powerful forward genetic screen to identify regulators of hematopoiesis.

Library Screening in Blood Differentiation

The ATF library was tested for morphological changes under conditions that provide extrinsic signals to differentiate to blood lineages (FIG. 23B) (3). A critical consideration to take when performing a forward genetic screen of this nature is to have a robust read-out of positive phenotypes (FIG. 23A). I relied on the expertise of the Slukvin lab to select for distinct morphological changes to blood cell types. Human H1 ES cells were treated with the ATF library, ATF library+TAL1, and ATF library+GATA2. Pluripotent cells were also transduced with lentivirus without an ORF (Empty) to account for false positive events induced by integration of a strong constitutive promoter upstream of a gene that induces hematopoiesis, disruption of a gene that represses hematopoiesis, or the cell's stress response to lentiviral infection. Among the treatments, cells treated with the ATF library+TAL1 differentiated into erythroblast-like cells and monocyte-like cells (FIG. 23C). Clusters of differentiated cells were expanded on methylcellulose, and expanded cells were pooled for collection.

Two pools of cells were sequenced for ATF identification: one of the erythroid-like phenotype and one of the monocyte-like phenotype. A combination of 4 ATFs were identified from the erythroid-like cells, and 15 ATFs were identified from the monocyte-like cells (FIG. 23C and Table 10). Because sequencing was not performed at a single cell level, when multiple ATFs were identified in the screen, it was not possible to determine the exact combinations that occurred simultaneously in a cell. Since fewer ATFs were identified for the erythroblast-like cells, the 4 ATFs identified from this phenotype were retested in the validation step (FIG. 23A). As a positive control, GATA2 and ETV2 (Condition 2) were overexpressed to induce differentiation to myeloid cells (FIG. 24) (3). TAL1 was expressed with each ATF individually and with an Empty control as a negative control. TAL1+ZFATF20 (Condition 6) generated cells of similar phenotype as the positive control. While TAL1+Empty (Condition 3) did not induce differentiation in the initial screen, in the validation step, the negative control did induce differentiation to endothelial-like cells, although their change in morphology was not as pronounced as with GATA2+ETV2 or Condition 6. TAL1+ATF5 (Condition 4) exhibited cells of a similar phenotype to those of TAL1+Empty. Conditions 5 and 7 did not yield any differentiated cells.

TABLE 10 Sequences of ATF Hits in Hematopoietic Differentiation ATF DNA sequence ZFATF1 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGTGATCGCCGCTTCTCCGTTTCC CACCGGCTCACCCATCACATCCGCATCCACACAGGCCAGA AGCCCTTCCAGTGCCGCATCTGCATGCGCAACTTCAGCCCT AGCAGAAGACTCACCCAGCACATCCGCACCCACACAGGCG AAAAGCCCTTCGCCTGCGACATCTGTGGAAGAAAGTTTGC CCAGAGCGAGGGGCGCAAGGTCCATACCAAGATCCACTTG CGGCAGAAGGACAAGAAAGCAGACAAAAGTGTTGTGGGG CGCGCCGACGCGCTGGACGATTTCGATCTCGACATGCTGG GTTCTGATGCCCTCGATGACTTTGACCTGGATATGTTGGGA AGCGACGCATTGGATGACTTTGATCTGGACATGCTCGGCT CCGATGCTCTGGACGATTTCGATCTCGATATGTTAATTAAC TACCCGTACGACGTTCCGGACTACGCTGGTTATCCCTATGA CGTCCCGGATTATGCAGGATCCTATCCATATGACGTTCCAG ATTACGCTTGA (SEQ ID NO: 72) ATF5 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGATTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCAGATCCCATCGGCCACCAGCCACATCCGCAT CCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCATG CGCAACTTCAGCGTCAGCCCGACCCTCACCCGACACATCC GCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCTG TGGAAGAAAGTTTGCCAGGAGCGACCAGCGCAAGAGACA TACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAGA CAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTTC GATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTGA CCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGAT CTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATCT CGATATGTTACCCGTACGACGTTCCGGACTACGCTGGTTAT CCCTATGACGTCCCGGATTATGCAGGATCCTATCCATATGA CGTTCCAGATTACGCTTGA (SEQ ID NO: 76) ZFATF6 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGTGATCGCCGCTTCTCCGTTTCC CACCGGCTCACCCATCACATCCGCATCCACACAGGCCAGA AGCCCTTCCAGTGCCGCATCTGCATGCGCAACTTCAGCCTC AGCCTAACCCTCACCAAACACATCCGCACCCACACAGGCG AAAAGCCCTTCGCCTGCGACATCTGTGGAAGAAAGTTTGC CAAAAGCAAACCACGCAAGACACATACCAAGATCCACTTG CGGCAGAAGGACAAGAAAGCAGACAAAAGTGTTGTGGGG CGCGCCGACGCGCTGGACGATTTCGATCTCGACATGCTGG GTTCTGATGCCCTCGATGACTTTGACCTGGATATGTTGGGA AGCGACGCATTGGATGACTTTGATCTGGACATGCTCGGCT CCGATGCTCTGGACGATTTCGATCTCGATATGTTAATTAAC TACCCGTACGACGTTCCGGACTACGCTGGTTATCCCTATGA CGTCCCGGATTATGCAGGATCCTATCCATATGACGTTCCAG ATTACGCTTGA (SEQ ID NO: 77) ZFATF10 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCGTATCCGACACTCTCACCGTACACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCGACAGCGATGAGCTCACCGATCACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCGAAAGCCGTAAACGCAAGGAAC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 81) ZFATF13 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGTGATCGCCGCTTCTCCGTTTCC CACCGGCTCACCCATCACATCCGCATCCACACAGGCCAGA AGCCCTTCCAGTGCCGCATCTGCATGCGCAACTTCAGCCCT AGCAGAAGACTCACCCAGCACATCCGCACCCACACAGGCG AAAAGCCCTTCGCCTGCGACATCTGTGGAAGAAAGTTTGC CAGGAGCGACCAGCGCAAGAGACATACCAAGATCCACTT GCGGCAGAAGGACAAGAAAGCAGACAAAAGTGTTGTGGG GCGCGCCGACGCGCTGGACGATTTCGATCTCGACATGCTG GGTTCTGATGCCCTCGATGACTTTGACCTGGATATGTTGGG AAGCGACGCATTGGATGACTTTGATCTGGACATGCTCGGC TCCGATGCTCTGGACGATTTCGATCTCGATATGTTAATTAA CTACCCGTACGACGTTCCGGACTACGCTGGTTATCCCTATG ACGTCCCGGATTATGCAGGATCCTATCCATATGACGTTCCA GATTACGCTTGA (SEQ ID NO: 92) ZFATF17 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCCGTTCCATCCGTCTCACCATACACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCGGTAGCGTTCCACTCACCATACACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCAGGAGCGACCAGCGCAAGAGAC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 96) ZFATF19 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCATATCCGGTGCGCTCACCGGGCACATCCGCA TCCACACAGGCCAGAAGCCCTTCGCCTGCGACATCTGTGG AAGAAAGTTTGCCAGGAGCGACCAGCGCAAGAGACATAC CAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAGACAA AAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTTCGAT CTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTGACCT GGATATGTTGGGAAGCGACGCATTGGATGACTTTGATCTG GACATGCTCGGCTCCGATGCTCTGGACGATTTCGATCTCGA TATGTTAATTAACTACCCGTACGACGTTCCGGACTACGCTG GTTATCCCTATGACGTCCCGGATTATGCAGGATCCTATCCA TATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 98) ZFATF20 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGATTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCCAGTCCGTGAATCTCACCGTGCACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCGCCAGCCCCCCGCTCACCATACACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCAGGAGCGACCAGCGCAAGAGAC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 99) ZFATF21 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGATTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCGTTTCCCACCGGCTCACCCATCACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCGTCAGCCCGACCCTCACCCGACACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCAGGAGCGACCAGCGCAAGAGAC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 100) ZFATF22 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCCGTTCCATCCGTCTCACCATACACATACGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACCTCAGCCACAGCGCTCATCTCACCCGACACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCAGGAGCGACCAGCGCAAGAGAC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 101) ZFATF23 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCGTTTCCCACCGGCTCACCCATCACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCCCTAGCAGAAGACTCACCCAGCACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCCAGAGCGAGGGGCGCAAGGTCC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 102) ZFATF24 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGTGATCGCCGCTTCTCCGTTTCC CACCGGCTCACCCATCACATCCGCATCCACACAGGCCAGA AGCCCTTCCAGTGCCGCATCTGCATGCGCAACTTCAGCGA CAGCGATGAGCTCACCGATCACATCCGCACCCACACAGGC GAAAAGCCCTTCGCCTGCGACATCTGTGGAAGAAAGTTTG CCAGGAGCGACCAGCGCAAGAGACATACCAAGATCCACTT GCGGCAGAAGGACAAGAAAGCAGACAAAAGTGTTGTGGG GCGCGCCGACGCGCTGGACGATTTCGATCTCGACATGCTG GGTTCTGATGCCCTCGATGACTTTGACCTGGATATGTTGGG AAGCGACGCATTGGATGACTTTGATCTGGACATGCTCGGC TCCGATGCTCTGGACGATTTCGATCTCGATATGTTAATTAA CTACCCGTACGACGTTCCGGACTACGCTGGTTATCCCTATG ACGTCCCGGATTATGCAGGATCCTATCCATATGACGTTCCA GATTACGCTTGA (SEQ ID NO: 103) ZFATF25 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCGTTTCCCACCGGCTCACCCATCACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCGACAGCGATGAGCTCACCGATCACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCCAGAGCGAGGGGCGCAAGGTCC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 104) ZFATF26 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGTGATCGCCGCTTCTCCGTTTCC CACCGGCTCACCCATCACATCCGCATCCACACAGGCCAGA AGCCCTTCCAGTGCCGCATCTGCATGCGCAACTTCAGCGTC AGCCCGACCCTCACCCGACACATCCGCACCCACACAGGCG AAAAGCCCTTCGCCTGCGACATCTGTGGAAGAAAGTTTGC CAGGAGCGACCAGCGCAAGAGACATACCAAGATCCACTT GCGGCAGAAGGACAAGAAAGCAGACAAAAGTGTTGTGGG GCGCGCCGACGCGCTGGACGATTTCGATCTCGACATGCTG GGTTCTGATGCCCTCGATGACTTTGACCTGGATATGTTGGG AAGCGACGCATTGGATGACTTTGATCTGGACATGCTCGGC TCCGATGCTCTGGACGATTTCGATCTCGATATGTTAATTAA CTACCCGTACGACGTTCCGGACTACGCTGGTTATCCCTATG ACGTCCCGGATTATGCAGGATCCTATCCATATGACGTTCCA GATTACGCTTGA (SEQ ID NO: 105) ZFATF27 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCGTATCCGACGCTCTCACCGTACACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCGTCAGCCCGACCCTCACCCGACACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCAGGAGCGACCAGCGCAAGAGAC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 106) ZFATF28 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGATTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCGTTTCCCACCGGCTCACCCATCACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCCTCAGCCTAACCCTCACCAAACACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCAAAAGCAAACCACGCAAGACAC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 107) ZFATF29 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCGTATCCGACGCTCTCACCGTACACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCGTCAGCCCGACCCTCACCCGACACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCGAAAGCCGTAAACGCAAGGAAC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 108) ZFATF30 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGATTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCGTATCCGACGCTCTCACCGTACACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCGACAGCGATGAGCTCACCGATCACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCGAAAGCCGTAAACGCAAGGAAC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 109) ZFATF31 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGATTGTGATCGCCGCTTCTCCGTTTCC CACCGGCTCACCCATCACATCCGCACCCACACAGGCGAAA AGCCCTTCGCCTGCGACATCTGTGGAAGAAAGTTTGCCAG AAGCATACACCGCAAGCGTCATACCAAGATCCACTTGCGG CAGAAGGACAAGAAAGCAGACAAAAGTGTTGTGGGGCGC GCCGACGCGCTGGACGATTTCGATCTCGACATGCTGGGTT CTGATGCCCTCGATGACTTTGACCTGGATATGTTGGGAAG CGACGCATTGGATGACTTTGATCTGGACATGCTCGGCTCC GATGCTCTGGACGATTTCGATCTCGATATGTTAATTAACTA CCCGTACGACGTTCCGGACTACGCTGGTTATCCCTATGACG TCCCGGATTATGCAGGATCCTATCCATATGACGTTCCAGAT TACGCTTGA (SEQ ID NO: 110)

ATF Targets

To identify the ATF target genes, cognate site identification (CSI) described in Chapter 3, was used to find the top 100 scoring motifs for each ATF (FIG. 25A). The sequence and energy landscapes (SELs) for the ATFs depict the comprehensive sequence specificity for each ATF (FIG. 25B). The locations of these top-scoring motifs were identified in the human genome, then annotated to the nearest genes. The frequency of occurrence in the genome was plotted as well as the frequency within ±1 kb of a transcription start site (TSS) (FIGS. 26A-26B). Although ZFATF19 (purple) had the most binding sites in the genome, ZFATF21 (brown) had the most binding sites near the TSS.

Discussion

This screen for morphological changes to blood lineages demonstrates that an ATF library can be used as a forward genetic screen to identify regulators of hematopoiesis. By testing the ATF library with TAL1, we were able to show that ATFs can promote differentiation into erythroid-like cells. Because ATF identification was not performed at the single cell level, we could not capture the combination of ATFs that induced the conversions to erythroid-like and monocyte-like cells. However, only four ATFs were expressed in the erythroid-like cells, so we were able to test them individually with TAL1 in the validation step. While TAL1 alone did not seem to induce differentiation in the initial screen, in the validation experiment, TAL1+Empty induced differentiation to endothelial-like cells. TAL1 has, indeed, been implicated in blood and endothelial development (4), although its capacity to differentiate pluripotent stem cells to blood lineages remains to be examined. Though morphologically distinct from TAL+ZFATF20, the propensity for TAL1+Empty to induce differentiation must also be further explored at the transcriptional level. In addition, chromatin-immunoprecipitation could be performed to elucidate the binding sites in cells. To better identify the combination of factors that induce differentiation into blood lineages, positive outcomes from future screens should be isolated as single cells for ATF identification. The results from this study suggests that the ATF library can be applied to other unanswered questions in the hematopoietic stem cell field.

Materials and Methods

Cell Culture:

Human H1 ES cells were grown in mTESR1™ medium (StemCell Technologies, Inc., Vancouver, CA) on Matrigel® substrate (BD Biosciences, NJ). For differentiation into blood lineages, cells were cultured in 3F media, composed of mTeSR basal media with FGF2 (20 ng/mL), SCF (100 ng/mL), and thrombopoietin (50 ng/mL). Expansion of cells with hematopoietic phenotypes was performed on methylcellulose. Cells were maintained in a humidified 37° C. incubator with 5% CO₂.

Lentivirus Production:

ATFs and the empty control were packaged into lentivirus using a second-generation lentiviral system. Lentivirus was produced in HEK293T cells by calcium phosphate transfection of pSIN expression, psPAX2 packaging, and pMD2.G envelope plasmids. Media containing virus was harvested 48-60 hours post-transfection. Lentivirus was centrifuged with a sucrose cushion at 25,000 rpm for 2 hours. Viral particles were suspended in PBS and concentrated virus was stored at −80° C. Viral titers were measured by counting HEK293T cells that survived selection after 2 days in media containing puromycin (3 μg/mL).

Lentiviral Transduction:

Lentiviruses were delivered to H1 cells with 8 μg/mL polybrene. Selection was performed in media containing puromycin (1 μg/mL first day, 1.5 μg/mL second day). The ATF was driven by a constitutive promoter, EF1α.

Identification of ATFs:

Cells exhibiting a phenotype of the hematopoietic lineage were picked an expanded on methylcellulose. Two-step nested PCR was performed from cell clusters after expansion. The first round of amplification was performed with ATF-forward-1 and ATF-reverse-1 primers (400 nM final concentration) in a 50 μL volume or 40 cycles. The second round of amplification was performed with 10 μL of the first PCR reaction, ATF-forward-2 and ATF-reverse-2 primers (400 nM final concentration) for 35 cycles. The second PCR reaction was purified and digested with AscI and NheI. The digested product was cloned into pcDNA 3.1 at the AscI and NheI restriction sites. Sanger sequencing was performed on the clones with the CMV-forward primer.

The number of sequences obtained to reach saturation was determined by calculating the probability that the chance of finding another unique ATF is less than 1%. (1−1/(n+1))^(x)<0.01, where n is the number of unique ATFs identified and x is the number of sequences needed.

Microscopy:

Micrographs were taken with a 10× objective on a Zeiss Observer. A1 with an AxioCam ICm1.

Cognate Site Identification:

HEK293 cells were transiently transfected with ATFs using Lipofectamine 2000 (Thermo Fisher #11668019). Cells that underwent a mock transfection (without an expression plasmid for ATFs) served as a negative control. Cells were harvested 48 hours post-transfection. Lysates were prepared by lysing 10⁷ cells in 300 μL of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, and 0.1% Na deoxycholate). A 21.5 G syringe needle was used for mechanical lysis. Lysates were centrifuged at 10 k×g for 10 min to separate precipitated genomic DNA from protein constituents. The supernatant, containing ATF protein, was used for CSI. HA magnetic beads (MBL #M132-9) were added to the cell lysates for immunoprecipitation. The binding reaction between the ATFs and 100 nM synthetic DNA was performed in binding buffer (25 mM HEPES, 80 mM KCl, 0.2 mM EDTA, 1 mM MgCl₂, 0.1 mM ZnSO₄) at 25° C. After pull-down, three quick washes with 100 μL ice-cold binding buffer were performed to remove unbound DNA. Magnetic beads were resuspended in a PCR master mix (Lucigen #30035-1) and the DNA was amplified for 15 or 18 cycles. Amplified DNA was column purified (Qiagen #28106), and this enriched DNA pool was used for the subsequent round of enrichment, for a total of three rounds. After three rounds of selection, Illumina sequencing adapters and a unique 6 bp barcode for multiplexing were added by PCR amplification. The starting library (Round 0) was also barcoded. Up to 180 samples were combined and sequenced in a single HiSeq2000 lane.

Cognate Site Identification Data Analysis:

Illumina sequencing yielded ˜180 million reads per lane. Reads were de-multiplexed by requiring an exact match to the 6 bp barcode and truncated to include only the 25 bp derived from the random portion of the library. On average, we obtained 709,300 reads per barcode. The occurrence of every k-mer (lengths 8 through 16 bp) was counted using a sliding window of size k. To correct for biases in our starting DNA library, we took the ratio of the counts of every k-mer to the expected number of counts in the mock-transfected control. The mock control was modeled using a 5th-order Markov Model derived from the sequencing reads corresponding to the starting library (Round 0). We then calculated a Z-score=(x−μ)/σ or (CSI score minus mean)/standard deviation for each k-mer, using the distribution of k-mer enrichment values (CSI score) for the ATF.

Bioinformatic Analysis of ATF Binding Sites:

The genomic locations of the top five or 100 high-scoring 10-bp motifs from CSI were identified in the mm10 genome by the findMotif utility from the UCSC Genome Browser. These genomic sites were annotated using Homer. Annotated sites were filtered to those within +1 kb of the TSS.

REFERENCES

-   1. Orkin S H, Zon L I (2008) Hematopoiesis: An Evolving Paradigm for     Stem Cell Biology. Cell 132(4):631-644. -   2. Doulatov S, Notta F, Laurenti E, Dick J E (2012) Hematopoiesis: a     human perspective. Cell Stem Cell 10(2):120-136. -   3. Elcheva I, et al. (2014) Direct induction of haematoendothelial     programs in human pluripotent stem cells by transcriptional     regulators. Nat Commun 5:4372. -   4. Bloor A J C, Sanchez M-J, Green A R, Göttgens B (2002) The role     of the stem cell leukemia (SCL) gene in hematopoietic and     endothelial lineage specification. J Hematother Stem Cell Res     11(2):195-206.

Example 4—Inducing Lytic Replication of Cytomegalovirus with ATFs

A majority of the population is infected with human cytomegalovirus (HCMV), a member of the β-herpesvirus family; however, the virus only exhibits pathogenic effects in immunocompromised individuals (1). Once cells are infected with HCMV, virions can either enter a latent state, in which a few viral genes are expressed, or enter a lytic state, in which the viral genes are robustly expressed, the viral genome is replicated, and new virions are produced to spread to other cells. The production of virions results in death of the host cell. It is remains unclear how long cells with HCMV in the latent state can survive. After latency is established, HCMV can reactivate, eliciting responses of the lytic state (1).

The exact mechanism controlling whether HCMV genes are expressed remains to be elucidated. Curiously, HCMV in pluripotent and hematopoietic stem cells establishes latency (2). The repressor protein Death Domain Associated Protein (DAXX) recruits histone deacetylases (HDACs) to the viral genome, and viral genes are silenced. In differentiated cells, the viral tegument protein, pp71, targets DAXX for degradation, and its degradation leads to expression of immediate early genes that facilitate viral replication in the lytic state. In undifferentiated cells, pp71 remains cytoplasmic. The cellular signals that regulate translation of pp71 to the nucleus are poorly understood.

We investigated the transcriptional mechanism that permits viral gene expression using an artificial transcription factor (ATF) library comprising 2.6×10⁶ ATFs, a complexity that encompasses 10-times the sequence space of all 9-bp permutations. An important and distinguishing feature of our ATF design is the incorporation of an interaction domain that allows two ATFs to dimerize and activate target genes in a synergistic manner. We tested the ATF library in permitting the expression of HCMV genes in monocytes where HCMV remains latent. Our results suggest that the ATF library can serve as a powerful forward genetic screen to identify regulators of lytic replication. Pinpointing the key nodes of a transcriptional network sufficient to license lytic replication would be therapeutic targets to prevent HCMV pathogenesis.

ATF Library Screening for Lytic Replication

We tested the ATF library in THP-1 cells, a human monocytic leukemia cell line, that has the potential to differentiate into macrophages (3). When HCMV infects THP-1 cells, viral genes are not expressed. Pretreatment with valproic acid (VPA), an HDAC inhibitor, followed by HCMV infection, results in the expression of viral proteins in about a quarter of the cell population. To test the ability of the ATF library to permit expression of viral genes, an HCMV strain AD169 expressing a fusion protein IE2-GFP (immediate early gene 2 fused to green fluorescent protein) was used to screen for lytic replication (FIG. 27B) (2).

For the ATF library screen, THP-1 cells were transduced with either the ATF library (multiplicity of infection=1) or lentiviruses lacking an open reading frame (Empty) but retaining the EF1a promoter and the puromycin selection gene. After selection of integration events with puromycin, cells were infected with HCMV and expression of the IE2-GFP reporter gene was assessed 20 hours later. Approximately, 0.8% of cells from the ATF-treated were GFP⁺, while only 0.5% of cells from the Empty control were GFP⁺ (FIG. 27C). The frequency of positive outcomes from the Empty control allows us to determine the rate of false positive events to expect out of the screen. For the HCMV screen, we expect a false positive rate of about 63% (⅝), which is quite high compared to other ATF library screens described in this thesis. Approximately 19% of cells pretreated with VPA followed by HCMV infection expressed IE2-GFP, and THP-1 cells uninfected with HCMV expressed no IE2-GFP. ATF-treated cells with the greatest GFP expression were isolated as single cells by cell sorting (FIGS. 27A and 27C). ATFs expressed in the sorted cells were identified by two-step nested PCR (FIG. 27D and Table 11). Each cell expressed one or two ATFs, one of which was common in four cells.

TABLE 11 Sequences of ATF Hits in HCMV Lytic Replication ATF DNA sequence ZFATF1 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGTGATCGCCGCTTCTCCGTTTCC CACCGGCTCACCCATCACATCCGCATCCACACAGGCCAGA AGCCCTTCCAGTGCCGCATCTGCATGCGCAACTTCAGCCCT AGCAGAAGACTCACCCAGCACATCCGCACCCACACAGGCG AAAAGCCCTTCGCCTGCGACATCTGTGGAAGAAAGTTTGC CCAGAGCGAGGGGCGCAAGGTCCATACCAAGATCCACTTG CGGCAGAAGGACAAGAAAGCAGACAAAAGTGTTGTGGGG CGCGCCGACGCGCTGGACGATTTCGATCTCGACATGCTGG GTTCTGATGCCCTCGATGACTTTGACCTGGATATGTTGGGA AGCGACGCATTGGATGACTTTGATCTGGACATGCTCGGCT CCGATGCTCTGGACGATTTCGATCTCGATATGTTAATTAAC TACCCGTACGACGTTCCGGACTACGCTGGTTATCCCTATGA CGTCCCGGATTATGCAGGATCCTATCCATATGACGTTCCAG ATTACGCTTGA (SEQ ID NO: 72) ZFATF2 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCGGGTCCGTAGTCCTCACCAAACACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCATAAGCACTGTTCTCACCGGTCACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCAACAGCGAGCCACGCAAGACCC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 73) ATF5 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGATTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCAGATCCCATCGGCCACCAGCCACATCCGCAT CCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCATG CGCAACTTCAGCGTCAGCCCGACCCTCACCCGACACATCC GCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCTG TGGAAGAAAGTTTGCCAGGAGCGACCAGCGCAAGAGACA TACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAGA CAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTTC GATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTGA CCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGAT CTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATCT CGATATGTTACCCGTACGACGTTCCGGACTACGCTGGTTAT CCCTATGACGTCCCGGATTATGCAGGATCCTATCCATATGA CGTTCCAGATTACGCTTGA (SEQ ID NO: 76) ZFATF6 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGTGATCGCCGCTTCTCCGTTTCC CACCGGCTCACCCATCACATCCGCATCCACACAGGCCAGA AGCCCTTCCAGTGCCGCATCTGCATGCGCAACTTCAGCCTC AGCCTAACCCTCACCAAACACATCCGCACCCACACAGGCG AAAAGCCCTTCGCCTGCGACATCTGTGGAAGAAAGTTTGC CAAAAGCAAACCACGCAAGACACATACCAAGATCCACTTG CGGCAGAAGGACAAGAAAGCAGACAAAAGTGTTGTGGGG CGCGCCGACGCGCTGGACGATTTCGATCTCGACATGCTGG GTTCTGATGCCCTCGATGACTTTGACCTGGATATGTTGGGA AGCGACGCATTGGATGACTTTGATCTGGACATGCTCGGCT CCGATGCTCTGGACGATTTCGATCTCGATATGTTAATTAAC TACCCGTACGACGTTCCGGACTACGCTGGTTATCCCTATGA CGTCCCGGATTATGCAGGATCCTATCCATATGACGTTCCAG ATTACGCTTGA (SEQ ID NO: 77) ZFATF7 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCCGTTCCATCCGTCTCACCATACACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCCCCAGCCCGCCACTCACCCCTCACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCCCAAGCCTTCTCCGCAAGCCACA TACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAGA CAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTTC GATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTGA CCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGAT CTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATCT CGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 78) ZFATF9 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCGGCTCCAATCGGCTCACCGAGCACATCCGC ATCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCTGTG GAAGAAAGTTTGCCAACAGCCCCCTACGCAAGGGTCATAC CAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAGACAA AAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTTCGAT CTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTGACCT GGATATGTTGGGAAGCGACGCATTGGATGACTTTGATCTG GACATGCTCGGCTCCGATGCTCTGGACGATTTCGATCTCGA TATGTTAATTAACTACCCGTACGACGTTCCGGACTACGCTG GTTATCCCTATGACGTCCCGGATTATGCAGGATCCTATCCA TATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 80) ZFATF23 ATGCATCCTATGAACAACCTGCTGAACTACGTGGTGCCGA AGATGAGACCTTACGCTTGCCCAGTGGAGTCCTGTGATCG CCGCTTCTCCGTTTCCCACCGGCTCACCCATCACATCCGCA TCCACACAGGCCAGAAGCCCTTCCAGTGCCGCATCTGCAT GCGCAACTTCAGCCCTAGCAGAAGACTCACCCAGCACATC CGCACCCACACAGGCGAAAAGCCCTTCGCCTGCGACATCT GTGGAAGAAAGTTTGCCCAGAGCGAGGGGCGCAAGGTCC ATACCAAGATCCACTTGCGGCAGAAGGACAAGAAAGCAG ACAAAAGTGTTGTGGGGCGCGCCGACGCGCTGGACGATTT CGATCTCGACATGCTGGGTTCTGATGCCCTCGATGACTTTG ACCTGGATATGTTGGGAAGCGACGCATTGGATGACTTTGA TCTGGACATGCTCGGCTCCGATGCTCTGGACGATTTCGATC TCGATATGTTAATTAACTACCCGTACGACGTTCCGGACTAC GCTGGTTATCCCTATGACGTCCCGGATTATGCAGGATCCTA TCCATATGACGTTCCAGATTACGCTTGA (SEQ ID NO: 102)

Validation of the ATFs from the screen showed minimal effects of the ATFs on IE2-GFP expression compared to the Empty control (FIG. 27E). It is important to note that the positive control (VPA) had fewer cells expressing IE2-GFP (8.4%) than in the original screen (19%/). Even the Empty control exhibited fewer cells expressing IE2-GFP (0.2% from 0.5%). This lower yield of IE2-GFP-expressing cells could be attributed to decreased levels of HCMV infection. HCMV must be sonicated to disaggregate the virions that tend to clump after freezing. A different sonicator was used for disaggregation in the validation step than in the original screen, resulting in different HCMV preparations. Experimental conditions could be optimized to increase HCMV infection for the purposes of revalidating the ATFs; however the high false positive rate suggests that the IE2-GFP system may not be a good system for ATF library testing.

ATF Targets and Mechanism of Action

To identify the ATF target genes, cognate site identification (CSI) described in Chapter 3, was used to find the top 100 scoring motifs for each ATF (FIG. 28A). The sequence and energy landscapes (SELs) for the ATFs depict the comprehensive sequence specificity for each ATF (FIG. 28B). The locations of these top-scoring motifs were identified in the human genome, then annotated to the nearest genes. The frequency of occurrence in the genome was plotted as well as the frequency within ±1 kb of a transcription start site (TSS) (FIGS. 29A-29B). ZFATF7 (brown ATF) appears to be highly specific, but white its preferred sites are highly represented in the human genome, they are less represented near the TSS.

Discussion

The HCMV screen for regulators permitting the expression of the viral genome to initiate productive lytic replication demonstrates that a system with a low false positive rate is critical for successfully identifying ATF hits. By comparing the number of cells that express IE2-GFP in the Empty control to the ATF library, it was apparent that the cell's response to lentiviral infection or the leaky nature of IE2-GFP expression makes it difficult to achieve a low false positive rate that is conducive to validating the ATF hits. While the validation step needs to be further optimized and replicated, this forward genetic screen demonstrated the challenge in testing an ATF library in a system where there may be false positives. The ATF library can either be tested in a different system to address the questions of licensing HCMV gene expression, or more cells must be sequenced for ATF validation, such that ATFs can be validated despite the high false positive rate.

Materials and Methods

Cell Culture:

THP-1 monocytes were grown in RPMI 1640 media supplemented with 10% FBS and 0.05 mM β-mercaptoethanol. Cells were maintained at a density of 2×10⁴-1×10⁶ cells/mL. Cells were maintained in a humidified 37° C. incubator with 5% CO₂.

Lentivirus Production:

ATFs and the empty control were packaged into lentivirus using a second-generation lentiviral system. Lentivirus was produced in HEK293T cells by calcium phosphate transfection of pSIN expression, psPAX2 packaging, and pMD2.G envelope plasmids. Media containing virus was harvested 48-60 hours post-transfection. Lentivirus was centrifuged with a sucrose cushion at 25,000 rpm for 2 hours. Viral particles were suspended in PBS and concentrated virus was stored at −80° C. Viral titers were measured by counting HEK293T cells that survived selection after 2 days in media containing puromycin (3 μg/mL).

Lentiviral Transduction:

Lentiviruses were delivered to THP-1 monocytes with 16 μg/mL polybrene by spinoculation. Cells were transduced with lentivirus at a density of 3×10⁵ cells/mL while spinning at 1000×g for 1 hour at 25° C. (MOI=1). Selection was performed in media containing puromycin (1 μg/mL). The ATF was driven by a constitutive promoter, EF1α.

HCMV Infection:

HCMV (AD169 strain) with the IE2-GFP reporter gene was thawed on ice and sonicated at 50% power 0.5 seconds on and 0.5 seconds off for 2×10 pulses. HCMV was placed on ice between sets of pulses. At a density of 8×10⁵ cells/mL, THP-1 cells were infected with HCMV for 1 hour at 37° C. with agitation every 15 min. For VPA pretreatment, cells were treated at a final concentration of 1 mM for 3 hours, then infected with HCMV.

Identification of ATFs:

Cells exhibiting a phenotype of the hematopoietic lineage were picked an expanded on methylcellulose. Two-step nested PCR was performed from cell clusters after expansion. The first round of amplification was performed with ATF-forward-1 and ATF-reverse-1 primers (400 nM final concentration) in a 50 μL volume or 40 cycles. The second round of amplification was performed with 10 μL of the first PCR reaction, ATF-forward-2 and ATF-reverse-2 primers (400 nM final concentration) for 35 cycles. The second PCR reaction was purified and digested with AscI and NheI. The digested product was cloned into pcDNA 3.1 at the AscI and NheI restriction sites. Sanger sequencing was performed on the clones with the CMV-forward primer.

The number of sequences obtained to reach saturation was determined by calculating the probability that the chance of finding another unique ATF is less than 1%. (1 −1/(n+1))^(x)<0.01, where n is the number of unique ATFs identified and x is the number of sequences needed.

Cognate Site Identification:

HEK293 cells were transiently transfected with ATFs using Lipofectamine 2000 (Thermo Fisher #11668019). Cells that underwent a mock transfection (without an expression plasmid for ATFs) served as a negative control. Cells were harvested 48 hours post-transfection. Lysates were prepared by lysing 10⁷ cells in 300 μL of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, and 0.1% Na deoxycholate). A 21.5 G syringe needle was used for mechanical lysis. Lysates were centrifuged at 10 k×g for 10 min to separate precipitated genomic DNA from protein constituents. The supernatant, containing ATF protein, was used for CSI. HA magnetic beads (MBL #M132-9) were added to the cell lysates for immunoprecipitation. The binding reaction between the ATFs and 100 nM synthetic DNA was performed in binding buffer (25 mM HEPES, 80 mM KCl, 0.2 mM EDTA, 1 mM MgCl₂, 0.1 mM ZnSO₄) at 25° C. After pull-down, three quick washes with 100 μL ice-cold binding buffer were performed to remove unbound DNA. Magnetic beads were resuspended in a PCR master mix (Lucigen #30035-1) and the DNA was amplified for 15 or 18 cycles. Amplified DNA was column purified (Qiagen #28106), and this enriched DNA pool was used for the subsequent round of enrichment, for a total of three rounds. After three rounds of selection, Illumina sequencing adapters and a unique 6 bp barcode for multiplexing were added by PCR amplification. The starting library (Round 0) was also barcoded. Up to 180 samples were combined and sequenced in a single HiSeq2000 lane.

Cognate Site Identification Data Analysis:

Illumina sequencing yielded ˜180 million reads per lane. Reads were de-multiplexed by requiring an exact match to the 6 bp barcode and truncated to include only the 25 bp derived from the random portion of the library. On average, we obtained 709,300 reads per barcode. The occurrence of every k-mer (lengths 8 through 16 bp) was counted using a sliding window of size k. To correct for biases in our starting DNA library, we took the ratio of the counts of every k-mer to the expected number of counts in the mock-transfected control. The mock control was modeled using a 5th-order Markov Model derived from the sequencing reads corresponding to the starting library (Round 0). We then calculated a Z-score=(x −μ)/σ or (CSI score minus mean)/standard deviation for each k-mer, using the distribution of k-mer enrichment values (CSI score) for the ATF.

Bioinformatic Analysis of ATF Binding Sites:

The genomic locations of the top five or 100 high-scoring 10-bp motifs from CSI were identified in the mm10 genome by the findMotif utility from the UCSC Genome Browser. These genomic sites were annotated using Homer. Annotated sites were filtered to those within +1 kb of the TSS.

REFERENCES

-   1. Penkert R R, Kalejta R F (2012) Tale of a tegument     transactivator: the past, present and future of human CMV pp71.     Future Virol 7(9):855-869. -   2. Penkert R R, Kalejta R F (2013) Human embryonic stem cell lines     model experimental human cytomegalovirus latency. MBio     4(3):e00298-13. -   3. Tsuchiya S, et al. (1980) Establishment and characterization of a     human acute monocytic leukemia cell line (THP-1). Int J Cancer     26(2):171-176.

The present invention has been presented by way of illustration and is not intended to be limited to the disclosed embodiments. Accordingly, those skilled in the art will realize that the invention is intended to encompass all modifications and alternative arrangements within the spirit and scope of the invention as set forth in the appended claims. 

We claim:
 1. An artificial transcription factor comprising a polydactyl zinc finger protein comprising two or three zinc finger domains, an interaction domain, and an activation domain, wherein each of the two or three zinc finger domains comprises a variable domain independently encoded by nucleic acid sequence of SEQ ID NO:2 (VNN-TCC-VNN-VNN-CTC-ACC-VNN), wherein each VNN of SEQ ID NO:2 is a codon corresponding to an amino acid selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V.
 2. The artificial transcription factor of claim 1, wherein the polydactyl zinc finger protein comprises three zinc finger domains encoded by SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, wherein each VNN of SEQ ID NOS:4, 5, and 6 is a codon corresponding to an amino acid selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V.
 3. The artificial transcription factor of claim 1, wherein the interaction domain comprises a 15-amino acid peptide that enables interaction with the hydrophobic face of the most 5′ zinc finger domain.
 4. The artificial transcription factor of claim 1, wherein the activation domain comprises four tandem repeats of DALDDFDLDML (SEQ ID NO:7).
 5. The artificial transcription factor of claim 1, further comprising a nuclear localization signal.
 6. The artificial transcription factor of claim 5, wherein the nuclear localization signal is encoded by an amino acid sequence selected from the group consisting of KDKKADKSVV (SEQ ID NO:11) and PKKKRKV (SEQ ID NO:12).
 7. A method of reprogramming a somatic cell to pluripotency, wherein the method comprises (a) exposing a somatic cell to a plurality of artificial transcription factors, wherein the artificial transcription factors are selected from the group consisting of ZFATF1, ZFATF2, ZFATF3, ZFATF4, and ZFATF5; (b) further exposing the somatic cell to a plurality of potency determining factors comprising Sox2, Klf4, and c-Myc; (c) culturing the exposed cells to obtain reprogrammed cells having a higher potency level than the somatic cell.
 8. The method of claim 7, wherein the plurality of ATFs comprises ZFATF1, ZFATF2, and ZFATF5.
 9. The method of claim 7, wherein the plurality of ATFs comprises ZFATF1, ZFATF2, and ZFATF4.
 10. The method of claim 7, wherein the plurality of ATFs comprises ZFATF1, ZFATF2, and ZFATF3.
 11. The method of claim 7, wherein the plurality of ATFs comprises ZFATF1 and ZFATF4.
 12. A method of directing differentiation of a pluripotent stem cell to a cardiomyocyte, wherein the method comprises (a) exposing a pluripotent stem cell to one or more artificial transcription factors (ATFs) selected from the group consisting of ZFATF1, ZFATF2, and ATF5; and (b) culturing the exposed cells of (a) in the presence of a Wnt activator for about 7-10 days, such that a population of cells comprising cardiomyocytes is obtained.
 13. The method of claim 12, wherein the ATFs comprise ZFATF1, ZFATF2, and ATF5.
 14. The method of claim 12, wherein the Wnt activator is a GSK3 inhibitor.
 15. The method of claim 14, wherein the GSK3 inhibitor is CHIR99021.
 16. A method of directing differentiation of a pluripotent stem cell to a hematopoietic lineage, wherein the method comprises (a) exposing a pluripotent stem cell to two or more artificial transcription factors (ATFs) selected from the group consisting of ZFATF19, ZFATF20, ZFATF21, ATF5, ZFATF1, ZFATF6, ZFATF10, ZFATF13, ZFATF17, ZFATF22, ZFATF23, ZFATF24, ZFATF25, ZFATF26, ZFATF27, ZFATF28, ZFATF29, ZFATF30, and ZFATF31; and (b) culturing the exposed cells of (a) in a basal culture medium comprising FGF2, SCF, and thrombopoietin, and in the presence of TAL1 for about 7-10 days, such that a cell population comprising hematopoietic lineage cells is obtained.
 17. The method of claim 16, wherein the ATFs comprise ZFATF19, ZFATF20, ZFATF21, and ATF5, and wherein the cell population comprises erythroid-like cells.
 18. The method of claim 16, wherein the ATFs comprise ZFATF1, ZFATF6, ZFATF10, ZFATF13, ZFATF17, ZFATF22, ZFATF23, ZFATF24, ZFATF25, ZFATF26, ZFATF27, ZFATF28, ZFATF29, ZFATF30, and ZFATF31, and wherein the cell population comprises monocyte-like cells.
 19. A method for preparing a zinc finger-based artificial transcription factor (ATF) library comprising a plurality of elements, whereby each element of said ATF library comprises a zinc finger backbone comprising two or three oligonucleotides, each encoding SEQ ID NO:3 (X⁻¹-S-X₂-X₃-L-T-X₆), an interaction domain, and an activation domain, wherein X⁻¹ is an amino acid preferably selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V; X₂ is preferably selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V; X₃ is preferably selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V; and X₆ is preferably selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V.
 20. The method of claim 19, wherein the ATF library has a complexity of about 2.62×10⁶.
 21. The method of claim 19, wherein the interaction domain comprises a 15-amino acid peptide that enables interaction with the hydrophobic face of the most 5′ zinc finger domain.
 22. The method of claim 19, wherein the activation domain comprises four tandem repeats of DALDDFDLDML (SEQ ID NO:7).
 23. The method of claim 19, further comprising a nuclear localization signal.
 24. The method of claim 23, wherein the nuclear localization signal is encoded by an amino acid sequence selected from the group consisting of KDKKADKSVV (SEQ ID NO: 11) and PKKKRKV (SEQ ID NO:12).
 25. A zinc finger-based artificial transcription factor (ATF) library obtained according to the method of claim
 19. 